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Introduction
The capacity to sense and respond to changes in temperature varies greatly between species. Many economically important crops such as cotton, soybean, maize and rice are chilling sensitive and unable to survive freezing temperatures (Larcher, 1995), while others like winter cereals, spinach and cabbage are able cope with temperatures below freezing. Plants can, in general, be classified into three broad categories based on their response to decreasing temperatures. The first category is the chilling sensitive plants, e.g. rice and maize, which are irreversible damaged by temperatures below 10-15°C. Plants that are chilling resistant show no dysfunction at temperatures above zero but are unable to develop freezing resistance. The freezing tolerant plants include plants that have an inherent ability to develop freezing tolerance. These plants are able to increase their tolerance by a process known as cold acclimation. Cold acclimation is induced by exposure to a period of low non-freezing temperatures that bring about genetic, morphological, and physiological changes, which results in the development of cold hardiness and the acquisition of freezing tolerance





1. Consequences of low temperature stress
1.1. Ice formation, dehydration and its effects on membranes
Low temperature injury can occur in all plants, but the mechanisms and types of damage vary considerably. Freezing injury is connected to the formation of ice crystals that occur once ice nucleation can no longer be avoided. The formation of ice is heavily dependent on the presence of ice nucleators, organic or inorganic substance that catalyse ice formation (Zachariassen and Kristiansen, 2000), and the cooling rate. It can to a certain extent, be prevented by the presence of solutes and by supercooling, allowing the cell fluid to be cooled down to temperature below freezing without ice nucleation. If the cooling rate is slow ice initiation occurs in the extracellular water, due to its lower solute concentration and higher levels of ice-nucleating agents. Without ice nucleators water remains in a supercooled state above -38°C, the temperature at which water self nucleates (Thomashow, 1998). In cold tolerant plants ice formation appear controlled, it starts at the outer surface of the cell wall and spreads through the extracellular areas, suggesting that ice nucleators are produced at specific sites (Brush et al., 1994). Potent ice nucleating agents have been found in the extracellular fluid of species ranging from trees such as Prunus, Citrus to annual plants like winter rye (Brush et al., 1994). As long as the plasma membrane remains intact the ice is confined to the
outside of the cell. Protrusion of ice into the cell is lethal, due to mechanical disruptions of the cell. The formation and containment is further influenced by antifreeze or thermal hysteresis proteins (Griffith et al., 1997; Pihakaski-Maunsbach et al., 2001). These proteins are able to decrease the temperature at which ice is formed in a solution without affecting the melting point of the solution. That there is no modification of the melting point implies that once bound to the ice nuclei they may inhibit the growth of the ice crystal even when in contact with supercooled water (Zachariassen and Kristiansen, 2000). In addition they affect the shape of the ice crystal and help inhibit recrystalisation. The formation of intercellular ice is also the major reason behind cellular dehydration at low temperature. The ice crystals decrease the water potential outside the cell and osmosis force water out, dehydrating the cell. As water is removed, the solute concentration increase and the likelihood of freezing is reduced. However, with continued growth of the ice, the cells become more desiccated, with profound effects on the cellular membranes, with the plasma membrane as the primary site of injury, as well as causing denaturation and precipitation of protein and molecules (Thomashow, 1998). Three types of freeze-induced membrane lesions have been characterized, depending on the stage of cold acclimation and the extent of freeze-induced dehydration (Umura et al., 1995). In non-acclimated plants the dehydration give rise to two different lesions associated with the plasma membrane. During freezing, osmotic contractions results in endocytotic vesiculation beneath the plasma membrane. Approximately 40% of the membrane surface area was lost this way in protoplasts of rye (Secale cereale) subjected to freezing (Dowgert and Steponkus, 1984), the reduction in area is irreversible and as a consequence the cell lyses when expansion occur during thawing. This common feature of non acclimated cells of rye, Arabidopsis and oat is referred to as expansion-induced lysis (Webb et al., 1994), and is associated with temperatures ranging from -2 to -4°C. When the temperature drops further, to below -5°C, and more severe dehydration occur, the injury is manifested as a complete loss of osmotic responsiveness during thawing. In non-acclimated tissue this is due to a phase transition of the phospholipids within the membrane from a lamellar (bilayer) to a hexagonal II (HII) phase when the water content of the cell drops to approximately 20%. During the HII-phase transition the phospholipids form long cylinders with the polar head groups orientated into the aqueous core rendering the membrane unable to expand (Umura et al., 1995).
1.2. Photosynthetic carbon metabolism
Photosynthesis is a highly regulated process transforming light energy into useable chemical energy in the form of ATP and NADPH for the assimilation of carbon and other essential nutrients. Proper function relies on a balance between the energy absorbed and trapped and the energy consumed by the metabolic reactions downstream. Low temperature has only a minor effect on the primary reactions of photosynthesis, catalysed by photosystem I and II, trapping the light energy and converting it into redox potential energy. A stronger effect can be seen on electron transport, increasing the viscosity of the thylakoid membrane, in addition to decreasing the rates of the enzymatic reactions of downstream metabolism (Huner et al., 1998). The balance between energy input through photochemistry and energy utilization through metabolism is called photostasis (Öquist and Huner, 2003) and since the temperature effects are skewed an imbalance is created. The shift in photostasis is further affected by the cessation in growth caused by low temperature inhibition of water and nutrient uptake. This disproportion may induce high excitation pressure and lead to photoinhibition of photosynthesis (Melis, 1999). The primary metabolic sink, consuming the chemical energy is represented by the reduction of CO2 to triose phosphate and the continuous regeneration of ribulose 1,5 bisphosphate (RuBP) in the Calvin cycle within the chloroplast. However, the assimilation of N and S also constitute sinks for photosynthetically generated redox *****alents and chemical potential energy (Paul and Foyer, 2001) and the effects of low temperature on these reactions are largely unknown. Optimal rates of photosynthesis are also dependent on a balance in carbon flow between the chloroplast and the cytosol. The most important pathway for end product synthesis in the cytosol is sucrose biosynthesis. If the rate of sucrose biosynthesis is running too fast, it will inhibit photosynthesis by withdrawing too many intermediates from the Calvin cycle and if it is too slow, it will inhibit photosynthesis by sequestering inorganic phosphor (Pi) in phosphorylated intermediates (Stitt et al., 1987). The coordinated regulation of the key-enzymes of sucrose biosynthesis fructose-1,6-bisphosphatase (cFBPase) and sucrose phosphate synthase (SPS) are necessary for adjusting the synthesis to the supply of carbon. Low temperature stress is known to cause a over-proportional repression of sucrose synthesis, the key regulatory enzymes together with UDP-glucose pyrophosphorylase are particularly sensitive. cFBPase converts fructose-1,6-bisphosphate to fructose-6-phosphate (F6P), and is inhibited by AMP and by the signal molecule fructose-2,6-bisphosphate (F2,6BP), which increase with decreasing levels of PGA and increasing levels of Pi. SPS situated downstream converts UDP-glucose and F6P to sucrose-6-phosphate and is regulated by glucose-6-phosphate (G6P) and Pi, and by phosphorylation (Huber and Huber, 1996). Phosphorylation of SPS results in a reduction in enzyme activity due to a decrease in the affinity for F6P and the activator G6P (Fig. 1).






The accumulation of soluble carbohydrates seen at low temperature cause inactivation of SPS by protein phosphorylation and the associated accumulation of fructose-6-phosphate (F6P) triggers the synthesis of fructose-2,6-bisphosphate, (Stitt, 1990). This inhibition of end product synthesis results in a build up of phosphorylated intermediates within hours after exposure to low temperature (Labate and Leegood, 1988), decreasing the Pi cycling between the cytosol and the chloroplast. This depletion of the stromal pool of Pi restricts the CF1-ATPase activity (Pammenter et al., 1993), hindering the synthesis of ATP needed for the regeneration of RuBP, thereby decreasing the rates of electron transport and contributing to down-regulation of photosynthesis (Foyer et al., 1990).
The accumulation of soluble carbohydrates further affects the photosynthetic capacity by repressing the expression of a number of genes encoding proteins important for photosynthesis. The addition of glucose to Chenopodium cell cultures or to spinach (Krapp and Stitt, 1995) caused a rapid decrease in mRNA levels of the small subunit of Rubisco, of chlorophyll a/b binding proteins and of the δ-subunit of the thylakoid ATPase. The change in gene expression resulted in a change in protein amount, 4-5 days after cold girdling for the amount of Rubisco had been reduced in half in leaves of tobacco, potato and spinach (Krapp and Stitt, 1995). These alterations were confirmed in Arabidopsis plants shifted to 5°C (Strand et al., 1997).
1.3. Respiration
Respiration is indispensable for the release of the metabolic energy bound by photosynthesis. Respiration is also vital as a source of carbon skeletons exported to the cytosol for growth and cellular maintenance (Fernie et al., 2004). Moreover, mitochondrial respiration is central for the continuance of photosynthetic activity, mostly due to the high energy requirement of sucrose biosynthesis. Respiration involves oxidation of highly reduced carbohydrates through glycolysis in the cytosol and the tricarboxylic acid (TCA) cycle within the matrix of the mitochondria, releasing CO2 and reducing *****alents NAD(P)H and FADH2. The NAD(P)H and FADH2 generated transfers their electrons to O2 via the mitochondrial electron transport chain, resulting in the consumption of oxygen and the release of ATP (Siedow and Day, 2000). Plant respiration is remarkably flexible with parallel glycolytic pathways in both the cytosol and plastid, alternative enzymes within glycolysis and the TCA cycle and the presence of both phosphorylating and non-ATP producing pathways of the electron transport chain (Rasmusson et al., 2004; Plaxton and Podesta, 2006). These features are suggested to be crucial aids for the acclimation to different stresses (McDonald and Vanlerberghe, 2006; Plaxton, 2006). Ultimately the equilibrium between photosynthesis and respiration is of great importance for the productivity and general function of the plant. Between 30 and 80% of the photosynthetically fixed carbon may be respired in the same day depending on species and growth conditions (Poorter and Navas, 2003). Respiration is known to be temperature sensitive. It is often assumed that the relationship between respiration and temperature is exponential with a constant Q10, i.e. proportional changes in respiration with a 10°C increase in temperature, typically around 2. However, it has been recognized that Q10 is not constant or near 2, except within a limited temperature range. Instead there is growing evidence that the thermal response varies among species and that it is dynamic, varying with the metabolic status of the plants, as well as acclimating to changes in temperature but also drought and light (Atkin et al., 2000; Bryla et al., 2001; Griffin et al., 2002). Changes in respiration as a function of temperature can be regulated by the supply of adenylates, and by the capacity of the enzymes of respiration. There is evidence that respiration capacity is limited at low temperature. At moderate temperatures (25°C) the addition of respiratory uncouplers and glucose is able to enhance the O2 uptake in intact roots of two species of Plantago, while it failed to trigger any response at low temperature (Covey-Crump et al., 2002). Furthermore, the addition ADP does not affect the substrate saturated O2 uptake in cold treated soybean cotyledons (Atkin et al., 2002). Finally, the fact that glycolytic substrates such as soluble sugars accumulates at low temperature suggests that respiratory flux is more likely to be controlled by the catalytic activity of the enzymes, either due to the inhibitory effect of low temperature on the activity per se and/or limitations in the enzymatic function.
2. Cold acclimation
Cold acclimation allows the plant to deal with the problems stated above. The process involves both avoidance from and tolerance to freezing (Fig.2). Freeze avoidance aim to lower the freezing temperature of the tissue, by supercooling along with the accumulation of soluble sugars, preventing ice from forming. Freezing tolerance allow ice to form without lethal outcome, and include changes that helps to protect the tissue. In order to cold acclimate the plant must first perceive the change in temperature, transduce the signal, activating or repressing appropriate genes to ultimately make the changes necessary.





2.1. Perception and cold signaling
An immediate effect of low temperature is a decrease in membrane fluidity and thus it has been implied that the cold sensor is connected to the plasma membrane. The first evidence came from a study in Synechosystis PCC6803 where chemically induced membrane rigidification caused the induction of a cold responsive gene (Los et al., 1993), a response that later was confirmed in plants by Orvar et al. (2000). Little is known about the actual cold sensors, and how the conversion of the physical signal is achieved. An early event in response to low temperature is the influx of calcium into the cytosol and the involvement of calcium in cold signaling has been demonstrated in numerous studies (Orvar et al., 2000). A rise in intracellular calcium has been observed within 10 seconds of cold treatment, inducing cold responsive genes such as RD29A (Nordin Henriksson and Trewavas, 2003). This induction of RD29A is reduced by the addition of calcium signaling antagonists, that also partially reduces the low temperature induced increase in calcium. The membrane rigidification correlated with the influx of calcium occurs through actin-filament reorganization. By treating Medicago sativa cells with membrane and actin microfilament stabilisers the influx of calcium and expression of cold-regulated (COR) genes could be prevented (Orvar et al., 2000). This rearrangement of the cytoskeleton may be responsible for the opening of integral stretch-sensitive calcium channels within the plasma membrane. Another important player suggested to be involved in cold signaling is the molecule inositol-1,4,5-triphosphate (IP3). Arabidopsis mutants (FRY1) with increased levels of IP3, show increased induction of low temperature and ABA responsive genes, indicating that it mediates ABA and stress signal transduction in plants. Exogenously applied IP3 results in a release of calcium from vacuolar vesicles and isolated vacuoles and mediates a transient increases in cytosolic calcium (Allen et al., 1995). These results indicate that the initial perception of abiotic stress results is dependent on both calcium and IP3. It is suggested that the specific calcium signature helps the cell distinguishing one stimulus from the other (Sanders et al., 1999). The changes in calcium levels results in alterations in protein phosphorylation within minutes after cold treatment (Monroy et al., 1998) suggesting that calcium acts as a secondary messenger in the signal transduction pathway, together with a MAP kinase cascade. Several protein kinases including the Arabidopsis MEKK1 are transcriptionally upregulated at low temperature (Mizoguchi et al., 1996), and the protein interacts with the downstream MAPKK MKK2 (Ichimura et al., 2000). Constitutively expressing or overexpressing MKK2 in Arabidopsis results in elevated MAPK kinase activity and improved freezing and salt tolerance, as well as a strong upregulation of several calmodulins and other calcium binding proteins (Teige et al., 2004).
2.2. Gene regulation and expression
Yamagochi-Shinozaki and Shinozaki (1994) identified a 9-bp DNA element in the promoter of the Arabidopsis RD29A gene. This regulatory element became known as the dehydration responsive element (DRE), containing the core sequence, CCGAC, designated the C-repeat (CRT). This regulatory sequence has since been found to be essential for low temperature responsiveness of several COR genes (Ouellet et al., 1998). Stockinger et al. (1997) isolated the first cDNA clone for a DRE/CRT-binding protein, named CBF1. Since then a number of CBFs has been isolated in Arabidopsis, the genes are referred to as DREB1A/CBF3, DREB1B /CBF1, DREB1C /CBF2. A fourth CBF paralog, CBF4, has been shown to be drought rather than cold responsive (Haake et al., 2002). The CBF-genes are induced within 15 minutes after exposure to low non-freezing temperatures followed by induction of genes that contain the DRE/CRT element. Constitutive expression of the CBF1, CBF3 and CBF4 genes in transgenic Arabidopsis plants results in the induction of COR gene expression and an increase in freezing tolerance without a low temperature stimulus (Haake et al., 2002). Similarly, overexpression of either AtCBF1 or AtCBF3 enhanced chilling and freezing tolerance in Brassica, tobacco and rice (Ito et al., 2006). Although the CBF regulon plays an important role in the regulation of the cold acclimation process, it is not the only participant. The eskimo-1 mutant in Arabidopsis is constitutively freezing tolerant without affecting the genes that belong to the CBF regulon, suggesting that there are other regulons important for the regulation of cold induced gene expression. Zhu et al. (2004) has reported the existents of such a pathway mediated by the HOS9 gene product. HOS9 encodes a transcription factor that controls at least 175 genes that does not appear to be regulated by the CBF regulon. Mutant hos9 plants also show an increased freezing sensitivity both before and after acclimation. It is clear that the subject is complex and that there are literally many paths to follow, much work is still needed to get a comprehensive understanding of cold sensing and signaling.
2.3. Responses increasing the cold tolerance
2.3.1. Stabilisation of membranes
An initial step in the cold acclimation process is to decrease the sensitivity of the plasma membrane. This is probably achieved both by changes in lipid composition, which alters the dehydration induced phase behavior of the membrane, and by the accumulation of substances in the surrounding cytosol, interacting with and stabilising the membrane. Following cold acclimation the proportion of practically every lipid component is altered, when expressed as mol % of the total lipid fraction (Lynch and Steponkus, 1987). The most pronounced changes are an increase in the proportion of phospholipids, due to an increase in the proportion of di-unsaturated types of phosphatidylcholine and phosphatidylethanolamine, and a decrease in the proportion of cerebrosides. These alterations changes the cryo behaviour of the plasma membrane and results in an increase in membrane fluidity (Steponkus et al., 1990). The importance of membrane fatty acid unsaturation has been confirmed, Arabidopsis mutants with reduced levels of polyunsaturated fatty acids were found to be more sensitive to chilling (Miquel et al., 1993). After cold acclimation the properties of the plasma membrane are altered with freeze-induced contractions resulting in exocytotic extrusions instead of endocytotic vesicles, these extrusions are reversibly incorporated into the membrane again during osmotic expansion. Loss of osmotic responsiveness still occurs in cold acclimated leaves but it is no longer associated with the transition to a HII-phase. Cold acclimated rye leaves have been subjected to temperatures as low as -35°C without the formation of HII-phase. Protoplasts from cold acclimated rye , oat and Arabidopsis has revealed deviations in the fracture plane (fracture-jump lesions) between the plasma membrane and various endomembranes in regions where these are brought into close contact and the occurrence of LOR was found to be connected to this phenomenon (Webb and Steponkus, 1993; Webb et al., 1994; Uemura et al., 1995). In addition to changes in lipid composition, there are experiments hinting at other mechanisms that potentially alter the cryobehaviour of membranes. Artus et al. (1996) showed that constitutive expression of COR15a, encoding a chloroplast targeted protein in Arabidopsis, enhances the in vivo freezing tolerance of chloroplasts in non acclimated plants by almost 2°C, it also had a positive effect on the in vitro freezing tolerance of protoplasts. The increase in freezing tolerance was assigned to an increase in membrane stability. In a following study Steponkus et al. (1998) demonstrated that the increased stability was a result of a decreased occurrence of freeze-induced lamellar-to-HII phase transitions. Furthermore, they showed that the protein encoded by COR15a, has the ability to increase the temperature at which the lamellar-to-HII phase transition occur and it also promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species of the chloroplast envelope. From these results the authors suggest that COR15a, push freeze-induced formation of the HII phase to lower temperatures by altering the intrinsic curvature of the inner membrane of the chloroplast envelope. This idea has recently been challenged by evidence localising the COR15a protein exclusively to the chloroplast stroma, raising the question of how the protein can affect the chloroplast envelope if it does not associate with the membrane. The protein was also shown to form oligomers which are capable of interacting with other proteins (Nakayama et al., 2007). The cryoprotective function of the protein was confirmed and there is still the possibility that COR15a work together with other membrane associated proteins that mediates the interaction. The authors, however, propose another function where the protein would activate enzymes or protect enzymes within the stroma from inactivation. This was supported by the fact that purified COR15a was able to protect L-lactate dehydrogenase against freeze-inactivation (Nakayama et al., 2007).
2.3.2. Readjustment of carbon metabolism
A well-known response to low temperature is an early and major shift in carbohydrate status of the plant, with both a quantitative and qualitative change in carbohydrate content. The most widespread accumulated free sugar at low temperature is sucrose (Guy et al., 1992). The increase in sucrose occurs in the cytosol rather than the vacuole (Koster and Lynch, 1992) and the increase can be as high as 10-fold. However, sucrose is far from the only solute accumulated, in higher plants, other commonly accumulated compatible solutes are raffinose, glucose, fructose, glycinebetaine and proline. It is not clear whether an increase in sugar content is causally related to the increase in freezing tolerance or if it is merely a low temperature response as there are very few studies of the cryoprotective role of sugars in planta. Nonetheless the ability to accumulate soluble sugars has been shown to be correlated with the capacity to cold harden in Arabidopsis wild type and transgenics with altered capacity for sucrose synthesis (Strand et al., 2003) and they have the ability to act as osmolytes, changing the osmotic potential of the cell and reducing the loss of water (Steponkus, 1984). Recent data has revealed that the early increase in sucrose in Arabidopsis in response to low temperature precedes the increase in transcription of one of the key enzymes involved in sucrose synthesis (SPS), showing that the initial build up of sucrose is not dependent on increased transcription (Kaplan et al., 2007). In addition, a number of in vitro studies indicate that these sugars have the power to stabilise membranes and proteins subjected to freezing or dehydration (Carpenter and Crowe, 1988; Umura et al., 2003). The increased concentration of sugars and other solutes has been hypothesised to increase the spatial separation between membranes by replacing the water. In vitro experiments has shown that the presence of mono- and disaccharides, in the intermembrane space can decrease the extent of which the membranes are brought close together, and thereby push the incidence of lamellar-to-gel phase to lower temperatures (Koster, 2001). Apoplastic sugars has also shown to decrease the adhesive energy that develops between hydrated plant surfaces and extracellular ice (Olien, 1992). Proline is a compatible solute that accumulates in response to a variety of environmental stresses. It is regarded as having multiple roles, acting as a mediator of osmotic adjustments, a stabiliser of subcellular structures, and a scavenger of free radicals but also as a buffer of cellular redox potential. Cold acclimation in wild-type Arabidopsis results in an up to 10-fold increase in proline (Strand et al., 2003).The Arabidopsis mutant eskimo-1, has been shown to be constitutively freezing-tolerant, these plants contain up to 30-fold more proline, 2-fold higher levels of total soluble sugars and a 3-fold higher increase in RAB18, a cold responsive gene encoding a dehydrin, in comparison to wild-type (Xin and Browse, 1998). It has also been shown that transgenic plants with suppressed degradation of proline are improved when it comes to freezing tolerance, supporting the results obtained with the eskimo-mutant (Nanjo et al., 1999). Proline is able to stabilise proteins during freezing in vitro by maintaining a hydration ****l of the protein in its native form (Carpenter et al., 1990). Many species also accumulate raffinose family oligosaccharides in response to low temperature. Similar to the other compatible solutes they help to stabilize the membranes by replacing the lost water, preventing lipid phase transitions (Hincha et al., 2003). Raffinose is synthesised from sucrose by the addition of galactose donated by galactinol, the synthesis of galactinol is considered to be the key regulatory step in the pathway, controlled by the enzyme galactinol synthase (GolS). In Arabidopsis, the increase in raffinose at low temperature has been associated with an increase in transcript (Liu et al., 1998) of one (GolS3) of the seven GolS genes (Taji et al., 2002; Kaplan et al., 2007). The induction is controlled by the CBF transcription factors (Fowler and Thomashow, 2002; Maruyama et al., 2004). Kaplan et al (2007) showed that the increase in raffinose was preceded by its substrates, as well as the transcriptional induction of two of the enzymes within the biosynthetic pathway, GolS3 and raffinose synthase (RS). The increase in transcription was shown to be an early event, peaking after 12h at 4°C after which the expression gradually declined. The increase in galactinol was detected the same time as the peak in expression of the enzymes and the increase in raffinose was seen after another 12h and the increase continued up until last sampling point (96h) when it had increased 45-fold (Kaplan et al., 2007).
However, the role of raffinose in enhancing freezing tolerance is contradictory. Transgenic Arabidopsis accumulating high levels of raffinose, due to an over-expression of one of the drought responsive GolS genes, increased the tolerance against drought, indicating a function in abiotic stress tolerance (Taji et al., 2002). Comparing two accessions of Arabidopsis (col-0 vs C24) with different freezing tolerance, showed that raffinose accumulated to a larger extent in the more tolerant accession (col-0) in both non-acclimated and acclimated state (Klotke et al., 2004). In support of this conclusion, transgenic petunia plants with reduced galactosidase activity, resulting in higher levels of raffinose has been shown to increase their freezing tolerance in comparison to wild-type plants (Pennycooke et al., 2003). On the other hand, Arabidopsis RS knockouts, which are unable to accumulate raffinose, and transgenic Arabidopsis plants with increased expression of GolS, accumulating higher amounts of raffinose in comparison to wild-type showed no difference in freezing tolerance in either their non-acclimated or acclimated state (Zuther et al., 2004). One explanation to this discrepancy is indicated by the fact that the increase in raffinose in the transgenic petunia is brought about through the manipulation of β-galactosidase, an enzymes that has been shown to have a variety of substrates besides the raffinose family oligosaccharides. Another possibility is that freezing tolerance in petunia is related to an increase in stachyose, which is not seen in Arabidopsis, rather than an effect of the raffinose accumulation.
There is still an ongoing debate about the causality between the accumulation of different soluble sugars and osmolytes and cold acclimation. In many cases there is still a lack of knowledge about the functional role and more direct studies beyond mere correlations are needed.
• Changes in the Calvin cycle
The Calvin cycle uses the energy captured in the electron transport chain to fix carbon from CO2, and convert it to organic compounds for further use by the organism. The cycle is autocatalytic, with each compound being both a substrate and a product, and comprised of three phases. In the first phase, carboxylation, CO2 is combined with a five carbon sugar, RuBP, yielding two molecules of 3PGA. The following step is the reduction of 3PGA to triose-phosphate by the enzymes phosphoglycerate kinase (PGK), NADP-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triose phosphate isomerase (TPI). The third phase, regeneration, refers to the regeneration of RuBP from triose phosphate, a process requiring several steps catalysed by aldolase (Ald), stromal fructose-1,6-bisphosphatase (sFBPase), transketolase (TK), seduhepthalose-1,7-bisophosphatase (SBPase), ribulose phosphate epimerase (R5P3ep), ribose phosphate isomerase (R5PI) and finally phosphoribulokinase (PRK). The cycle needs to be tightly regulated in order to prevent the depletion of ATP and NADPH and intermediate pools, this is done through enzymatic control. Rubisco, sFBPase, SBPase and PRK all catalyse irreversible reaction, in addition these enzymes together with GAPDH are highly regulated by light dependent changes in the chloroplast such as reduced thioredoxin, pH and magnesium concentration.
The recovery of photosynthetic capacity in cold acclimated plants is dependent on an increase in the activity of the enzymes within the Calvin cycle. The cold response of eight of the enzymes has been examined in Arabidopsis and after 10 days at 5°C only three of the enzymes showed small increases in activity (Strand et al., 1999). However, in leaves that had been allowed to develop at 5°C all of the enzymes showed an increased, with Rubisco, sFBPase, SBPase, GAPDH and Ald exhibiting at least a doubling in activity. The increase was not due to a specific increase in total activity but rather to a general increase in the total leaf protein that results in an increased activity. A recent study of the chloroplast stromal proteomes of Arabidopsis revealed that there was an increase in abundance, on a plastid protein basis, of Rubisco in response to low temperature, while PGK, GAPDH, sFBPase, R5P3ep and PRK showed a significant reduction relative to the total pool of proteins (Goulas et al., 2006) in agreement with previous result demonstrating changes in enzymatic capacity on a per leaf protein basis (Strand et al., 1999). GAPDH, Ald, sFBPase and SBPase are all situated directly downstream of Rubisco and their products serve as precursors for sucrose and starch biosynthesis, as well as the regeneration of RuBP. An up regulation of the reduction phase supports carbon fixation and end product synthesis, at same time as lower relative activity of the regeneration phase restricts fluxes and decrease sequestering of Pi in metabolites of the regenerative parts. In contrast to Arabidopsis, spring cultivars of wheat and Brassica napus, with restricted ability to cold acclimate, exhibit limited capacity to increase the activity of the enzymes involved in carbon metabolism (Hurry, 1995). Correlated with the enhancement of certain Calvin cycle enzymes in cold developed leaves is a release in suppression of photosynthetic gene expression in Arabidopsis (Strand et al., 1997). These cold developed leaves showed a full recovery of the transcript levels even though they maintain high amounts of soluble carbohydrates. It has, however, been shown that sugar accumulation per se is not the signal regulating the expression of photosynthetic genes but the signal may be related to the metabolism of hexose phosphates (Krapp et al., 1993; Jang and Sheen, 1994; Krapp and Stitt, 1995).
• Changes involving sucrose biosynthesis
The strong inhibition of sucrose synthesis plays an important role in the cold induced loss of photosynthetic activity. The inhibition imposed on sucrose synthesis by low temperatures is counteracted during cold acclimation by a fast and strong increase in both the activity and amount of cFBPase, SPS and UGPase (Guy et al., 1992; Hurry et al., 1994; Hurry et al., 1995; Strand et al., 1997; Strand et al., 1999; Ciereszko et al., 2001). Arabidopsis leaves that develop at 5°C show a four to five-fold increase in the activity of both cFBPase and SPS, as well as an increase in expression of the genes, supporting the increase in protein (Strand et al., 1997; Strand et al., 1999). The strong increase in activity of these enzymes has also been demonstrated in other cold hardy herbaceous plants such as spinach, oilseed rape, winter wheat and winter rye, supporting the notion that this is a central feature of the cold acclimation process (Guy et al., 1992; Hurry et al., 1994; Hurry et al., 1995). In leaves of Arabidopsis the development of freezing tolerance is connected to a shift in the partitioning of newly fixed carbon into soluble carbohydrates rather than starch (Strand et al., 1997; Strand et al., 1999; Hurry et al., 2002; Strand et al., 2003; Lundmark et al., 2006), demonstrating importance of the specific increase of the sucrose biosynthesis pathway. The enhancement of sucrose biosynthesis serves to increase the rates of Pi release and reduce the Pi-limitation of photophosphorylation. Moreover, there are studies linking SPS activity and the rate of sucrose export, suggesting that the up-regulation of sucrose synthesis may be of importance for re-establishing sucrose export to developing sink tissues (Baxter et al., 2003). Arabidopsis plants with decreased expression of cFBPase have altered carbon metabolism with a switch from sucrose to starch synthesis as a consequence, resulting in decreased leaf sugar levels during the day that rise at the end of the night due to starch breakdown. In total the decreased expression of cFBPase results in a metabolic profile that is diagnostic of a stimulation of starch synthesis and inhibition of photosynthesis due to decreased recycling of Pi, comparable to what happens at low temperature (Strand et al., 2000). These plants have limited capacity to cold acclimate, almost certainly as a consequence of their inability to enhance sucrose biosynthesis. Wild-type Arabidopsis was shown to increase their freezing tolerance down to -7°C after exposure to 5°C for a period of 10 days, whereas the cFBPase antisense plants only reached a freezing tolerance of about -5°C (Strand et al., 2003). Further evidence for the importance of the up-regulation of sucrose biosynthesis is provided by transgenic Arabidopsis plants over expressing maize SPS. These plants have higher flux of carbon into soluble sugars and a significant increase in soluble sugar/starch ratio, they also show significantly less inhibition of photosynthesis when shifted to low temperature (Strand et al., 2003). This improvement of sucrose synthesis resulted in an increased freezing tolerance, reaching -9°C after 10 days at 5°C (Strand et al., 2003).
• Redistribution of Pi
Redistribution of inorganic phosphate is important for the recovery of sucrose synthesis. In warm grown leaves of well-fertilized plants cytosolic Pi pool are relatively constant and excess Pi is located within the vacuole (Bieleski and Ferguson, 1983; Foyer and Spencer, 1986; Sharkey and Vanderveer, 1989). The depletion of the Pi-pool at low temperature (Hurry et al., 1994; Hurry et al., 1995; Strand et al., 1999) cause a high demand for accessible Pi in the cytoplasm. Although the total pool of Pi within the cells do not change greatly after cold exposure there are indications of a redistribution from the vacuole towards the cytosol (Strand et al., 1999), increasing the cytosolic Pi pool in the long-term. This indicates that Pi limitation of photosynthesis is not likely to occur after long-term exposure to low temperature. Studies of Arabidopsis mutants pho1-2 and pho2-1 with decreased and increased leaf phosphate levels respectively provide evidence or the importance of Pi concentration in the development of cold acclimation, related to photosynthetic carbon metabolism. (Hurry et al., 2000). In cold developed leaves of pho1-2 the levels of accessible Pi increased several fold and was connected to an increased induction of SPS activity and SPS and cFBPase expression compared to WT, whereas no increase could be detected in pho2-1. The repression of photosynthetic genes, after low temperature exposure, was abolished in the pho1-2 mutant and heightened in the pho2-1 mutant. In all cold acclimation was improved in pho1-2 and weakened in the pho2-1 mutant compared to WT.
• Changes in the inter-conversion between starch and sucrose
The interconversion of starch to sucrose has received considerable recent attention in connection with the cold-induced sugar accumulation that is believed to enhance the degree of freezing tolerance. The exact molecular mechanism behind the accumulation, especially during an early phase of cold acclimation is unclear. However the conversion of starch into soluble sugars during temperature stress has been a well known fact for a long time (Siminovitch et al., 1952; Parker, 1962; Sakai, 1974). The importance of starch breakdown during early stages of cold acclimation was recently demonstrated using Arabidopsis mutants called the ******1 (starch excess1) mutants (Caspar et al., 1991; Yu et al., 2001). ******1 has been shown to encode a starch-related α-glucan/water dikinase (GWD), a protein that facilitates the phosphorylation of α 1􀃆4 glucan chains at the C6 and C3 position (Ritte et al., 2002). Studies in potato and Arabidopsis have shown that GWD acts as a global regulator of starch catabolism. The six allelic ******1 mutants in Arabidopsis all show impaired starch degradation in dark-adapted leaves (Caspar et al., 1991; Yu et al., 2001), connected to reduced starch phosphorylation levels (Yu et al., 2001), similar to the results of GWD antisense potato plants (Lorberth et al., 1998). The ******1 mutants display reduced ability to increase their freezing tolerance within the first 24h of cold exposure (Yano et al., 2005). They fail to accumulate maltooligosaccharides, as well as normal levels of Glc and Fru during the early stages of cold acclimation, but do not show any abnormal phenotypes in the fully cold acclimated state (Yano et al., 2005). A second GWD-like protein (GWD3 or phosphoglucan water dikinase (PWD)) has been discovered in Arabidopsis. This enzyme catalyses the same reaction as GWD, and is required for normal starch breakdown (Baunsgaard et al., 2005; Kotting et al., 2005). Unlike GWD, GWD3 does not act on unphosphorylated glucans, suggesting that it acts downstream of GWD (Baunsgaard et al., 2005). Although the mechanism behind the degradation of the starch granule is not fully elucidated, it is clear that phosphorylation of starch is essential for hydrolytic breakdown to take place. It is possible that the α-glucan/water dikinases phosphorylates the amylopectin making it accessible for the degrading enzymes (Blennow et al., 2002; Ritte et al., 2002). In the chloroplast there are two alternative pathways of further degradation. First, chloroplastic glucan phosphorylase (PHS1) (Zeeman et al., 1998) can catalyse the conversion of terminal glucosyl units to glucose-1-phosphate, which can be converted into triose phosphate and exported out of the chloroplast via the triose phosphate translocator (Hausler et al., 1998). The second possibility is that β-amylases hydrolyses the β-1,4-glycosidic linkages of the polyglucan chains at the non-reducing end producing maltose or maltotriose, which are too short to be further metabolised by the β-amylases. There are solid evidence that maltotriose is further metabolised by the disproportionating enzyme 1 (DPE1). In dpe1 mutants of Arabidopsis, maltotriose accumulated in the dark and starch breakdown was retarded (Critchley et al., 2001) DPE1 produces glucose and maltopentose. The glucose residues can be exported out to the chloroplast via the glucose transporter while the maltopentose can be attacked again by β-amylase, producing maltose and maltotriose. In the end the net products of the breakdown of these linear oligosaccharides would be maltose and to some extent glucose. Studies have shown that degradation of linear glucans in Arabidopsis usually takes place via BMYs rather than PHS1. Knockout mutants of PHS1 have normal rates of starch breakdown (Zeeman et al., 2004) while knockouts of one of the chloroplastic BMYs have reduced rates of degradation (Smith et al., 2004; Kaplan and Guy, 2005), similar to what is seen in BMY antisense plants of potato (Scheidig et al., 2002). Arabidopsis has nine BMY genes, including one that has been targeted (BMY8) (Ferreira et al., 2004), and three that are predicted to be targeted, to the chloroplast (BMY6,7,9) (Scheidig et al., 2002). Numerous studies have shown that maltose is exported out of isolated chloroplasts (Servaites and Geiger, 2002; Weise et al., 2004) and the discovery of a maltose transporter located in the chloroplast envelope (Niittyla et al., 2004) brought further attention to this route of carbon export to the cytosol.
Once in the cytosol maltose is proposed to be further metabolised by the disproportionating enzyme 2 (DPE2) to glucose and/or sucrose, and maltodextrins (Chia et al., 2004; Lu and Sharkey, 2004). DPE2 deficient plants show up to a 100-fold increase of maltose in the leaves, together with lower night-time levels of sucrose (Chia et al., 2004; Lu and Sharkey, 2004). The DPE2 reaction is thus likely to be essential in cytosolic maltose metabolism. Under in vitro conditions DPE2 transfers glycosyl residues to glycogen, using maltose as the glycosyl donor (Chia et al., 2004). No glycogen-like glucan has been found in the cytosolic compartment of plant cells, so it is likely that DPE2 uses this highly branched homoglucan as a substitute for an endogenous carbohydrate not yet identified. Similar to DPE2 the cytosolic phosphorylase (PHS2) shows strong affinity towards glycogen. It is possible that these enzymes use the recently isolated water soluble heteroglucans (SHG) as substrates. The most prominent constituents of these SHGs are arabinose, galactose and glucose, their pattern of glycosidic linkages is highly complex with more than 20 different linkages. Both low- and high-molecular weight heteroglucans has been found within the cytosol of mesophyll cells. In vitro assays have revealed that heteroglucans isolated from leaves acts as acceptor for the both the DPE2 and the PHS2 catalysed glycosyl transfer reaction (Fettke et al., 2005; Fettke et al., 2005; Fettke et al., 2006). DPE2 deficient Arabidopsis mutants have been shown to have unchanged cytosolic heteroglucan pools during the light/dark cycle while the wild type pool increased in size in the dark (Fettke et al., 2006).
The structure of the heteroglucans differed, with the mutants having higher glucosyl content, especially in the outer chains accessible to a hydrolytic or phosphorolytic attack. It has been demonstrated that recombinant DPE2 uses maltose in preference to other oligosaccharides in the presents of SHG or glycogen. β-amylase transcript and/or activity is induced during low temperature stress and connected to an increase in maltose content (Nielsen et al., 1997; Kaplan and Guy, 2004). Shifting Arabidopsis to low temperature results in an increased expression of one of the chloroplastic β-amylase (BMY8), the increase occur as early as 2h after exposure to cold (Seki et al., 2001) and after 12h the expression had increased 14 fold (Sung et al., 2001). The increase in maltose in Arabidopsis was shown to peak after 4h at 4°C, while the transcript levels of BMY8 peaked after 24h, suggesting that the increase in maltose during cold shock conditions is not exclusively caused by increased transcription. Other contributing factors could be increased β-amylase activity and/or decreased DPE2 or MEX1 activity. Maltose has been shown, in in vitro assays, to have the ability to protect proteins and the photosynthetic electron transport chain under freezing stress (Kaplan and Guy, 2004).
• Changes in respiration
There are numerous physiological studies demonstrating respiratory acclimation in response to a new temperature regime (Atkin et al., 2000; 2002; Bolstad et al., 2003). The potential for thermal acclimation of respiration varies among species and across different populations within species and is suggested to be, to some extent, dependent on the environment to which the plant is adapted (genetically based) (Larigauderie and Körner, 1995; Arnone and Körner, 1997; Tjoelker et al., 1999; Atkin et al., 2000; Loveys et al., 2003). Larigauderie and Körner (1995) found growth at low temperature to result in little or no acclimation in a number of alpine and lowland species, while a number of other species, of the same genera, acclimated. It is unclear whether there are systematic differences in temperature acclimation potential among species of different functional groups, however six of the genera mentioned above showed differences in ability to acclimate. A number of broad-leaved tree species have been shown to have a lower capacity to acclimate when compared to a number of conifers (Tjoelker et al., 1999), suggesting that functional traits might be used to predict the ability to acclimate.
Acclimation to lower temperatures consistently results in increased respiration rates, in comparison to warm grown plants, when measured at a common temperature (Rook, 1969; Körner and Larcher, 1988; Arnone and Körner, 1997; Atkin et al., 2000; Covey-Crump et al., 2002). As with photosynthesis, there is a developmental aspect and two different stages of respiratory acclimation has been identified by Atkin and Tjoelker (2003). The first stage, referred to as type I acclimation, represents fast adjustments that takes place in pre-existing tissues within days following a sustained change in temperature. This type of acclimation relies on alterations in the cellular machinery already in place and reflects a change in the availability of respiration substrate and/ or the degree of adenylate restriction of respiration (Atkin et al., 2000). The second stage, type II acclimation, is changes seen in tissues that develop at the new temperature. As with full acclimation of photosynthesis complete acclimation of respiration appear to require the development of new tissue with altered morphology and biochemistry (Atkin and Tjoelker, 2003). Leaves that develop at a new lower temperature have higher respiration rates in comparison to warm grown plants over a wide range of 3). It has also been shown that cold developed leaves of Arabidopsis have higher leaf mass area and higher nitrogen and protein concentration than leaves that developed at warm temperatures (Strand et al., 1997; Strand et al., 1999; Tjoelker et al., 1999), this could lead to an increase in the total amount of proteins invested in the respiratory chain. The cold developed leaves of Arabidopsis exhibit increased mitochondria density in epidermal cells and an increase in the cristae to matrix ratio in mesophyll cells, changes associated with an increase in respiratory capacity and respiratory rate (Armstrong et al., 2006). Temperature often has a greater relative affect on the rate of substrate use, by growth, maintenance processes and respiration in it self, than substrate production, altering the steady-state concentration of respiratory substrate. At low temperature this usually results in an increase in soluble sugars in source leaves (Strand et al., 2003; Lundmark et al., 2006), and thus an increased substrate availability for respiration. Although studies have not found sugar levels to be important in respiratory acclimation (Atkin et al., 2000; Talts et al., 2004), changes in sugar concentration may still influence acclimation by affecting gene expression (Sheen, 1994; Koch, 1996). 01025Cold (Type II)Cold (Type I)WarmMeasuring temperature (°C)Respiration
A Like the case with substrates the demand for ATP can be reduced in the cold, resulting in an inadequate supply of ADP, at the same time as the potential for ATP synthesis is limited due to changes in enzymes capacity (Atkin and Tjoelker, 2003). For example Ribas-Carbó et al. (2000) found the flux through the alternative oxidase (AOX) pathway to increase in pre-existing tissues after several days of exposure to low temperature, possibly to ensure that the TCA cycle remains active measuring temperatures (Atkin and Tjoelker, 200 cclimation of respiration may be linked to the demand and synthesis of ATP. under conditions of low ATP demand (Atkin et al., 2005). A third factor that might affect acclimation of respiration is the levels of reactive oxygen species (ROS) (Atkin et al., 2005). Low temperature potentially increases the amount of ROS due to over reduction of the electron transport chain in both chloroplasts and mitochondria (Purvis and Shewfelt, 1993; Purvis, 1997; Møller, 2001; Foyer and Noctor, 2003). ROS in itself is a powerful signaling agent (Wagner, 1995; Karpinski et al., 1999; Foyer, 1997) and could induce pathways causing an increase in enzymes that eventually results in a reduction in ROS, such as AOX (Purvis and Shewfelt, 1993; Wagner and Krab, 1995; Møller, 2001). Overexpressing AOX in tobacco cells reduced the amount of ROS in half, while antisense inhibition increased the production (Maxwell et al., 1999). In support, exposure to low temperature has been reported to result in an increase in AOX protein and/or activity (Gonzalez-Meler
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