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A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis
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A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis Mark Stitt* and Vaughan Hurry† Low temperatures lead to the inhibition of sucrose synthesis and photosynthesis. The biochemical and physiological adaptations of plants to low temperatures include the posttranslational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in the leaf protein content. Recent progress has been made in understanding both the signals that trigger these processes and how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation. Addresses *Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany; e-mail: [email protected] †Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Sweden Correspondence: Mark Stitt Current Opinion in Plant Biology 2002, 5:199–206 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 21 March 2002 Abbreviations ABA abscisic acid CBF CRT-binding factor cFBPase cytosolic fructose-1,6-bisphosphatase COR COLD REGULATED CRT C-repeat desB fatty acid desaturaseB DRE dehydration-responsive element DREB1 DRE-binding protein1 Pi inorganic orthophosphate Ru1,5bisP ribulose-1,5-bisphosphate sfr4 sensitive-to-freezing4 SPS sucrose phosphate synthase
Introduction Low temperature is one of the most important factors affecting plant performance and distribution [1,2]. At high latitudes or altitudes, the problem of coping with low temperatures is exacerbated by the need to prolong the growing season beyond the short summer. Low temperatures slow down enzyme-catalysed reactions and modify the conformation of lipids and other macromolecules, with consequences for most biological processes. It is difficult to determine which processes are affected most severely by cold, and differential responses generate complex indirect effects. Sub-freezing temperatures lead to severe damage caused by dehydration when ice forms outside the cell or, in extreme cases, to freezing of the cytoplasm. Many species, including crops such as maize, bean, tomato and potato, have only a limited capacity to cope with low
temperatures [2]. Cold-hardy herbaceous species, including Arabidopsis [3,4,5••] and crops such as winter cereals, winter rape, spinach and cabbage [6–9], grow at low temperatures and survive freezing temperatures. Biennials and woody perennials that are adapted to high latitudes cope with temperatures down to –40°C or lower [2]. Low temperature tolerance develops in cold-hardy species during a period of exposure to low but non-freezing temperatures in a multifacetted process termed cold-acclimation [10•]. In this review, we discuss the biochemical and physiological adaptations of plants to low temperature. These include the post-translational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in leaf protein content. We then discuss signals that may trigger these processes and consider how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation.
An important role for sugars in cold acclimation Descriptive ecological and agronomic studies have uncovered a strong correlation between sugar concentrations and frost resistance [2,7,11–14]. Likewise, changes of light regime during cold acclimation revealed a strong correlation between sugar levels and frost tolerance in Arabidopsis [15]. The sugar concentrations in transformants with antisense inhibition of cytosolic fructose-1,6-bisphosphatase (cFBPase) and sucrose phosphate synthase (SPS) expression, or with overexpression of maize SPS, correlated with the extent of frost tolerance after cold acclimation [16]. Similarly, the constitutive increases in the frost tolerance of the eskimo1 mutant [17] and in transformants over-expressing the C-repeat (CRT)-binding factor (CBF)/dehydration-responsive element (DRE)-binding protein1 (DREB1) [18••] correlated with increased sugar contents. The sensitive-to-freezing4 (sfr4) mutant is impaired in cold acclimation and has sugar levels that are lower than those of wildtype Arabidopsis plants [19,20]. Recent research has started to define the functional importance of sugar metabolism during cold acclimation. Progress has been achieved by investigating which adaptations in photosynthetic carbon metabolism provide increased sugar levels, which signals are involved, and how sugar metabolism interacts with other responses during cold acclimation.
An over-proportional inhibition of sucrose synthesis reduces photosynthesis at low temperatures During photosynthesis, CO2 combines with ribulose-1,5bisphosphate (Ru1,5bisP) to form glycerate-3-phosphate, which is reduced to triose-phosphate using NADPH and
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ATP that is generated by photosynthetic electron transport. The majority of the triose phosphates are retained in the chloroplast to regenerate Ru1,5bisP. The surplus is converted to end products, releasing inorganic orthophosphate (Pi) that recombines with ADP to regenerate ATP. The most important pathway for end-product synthesis involves the export of triose phosphates to the cytosol, where they are converted to sucrose via cFBPase and SPS. Optimal rates of photosynthesis require an appropriate balance between the rates of carbon fixation and sucrose synthesis [21–23]. Excessive sucrose synthesis depletes the phosphorylated Calvin cycle intermediates and inhibits the regeneration of Ru1,5bisP. Conversely, inadequate sucrose synthesis leads to accumulation of phosphorylated intermediates and depletion of Pi, resulting in inhibition of ATP synthesis, accumulation of glycerate3-phosphate and inactivation of Rubisco. Studies performed in the late 1980s with barley and spinach showed that falling temperatures lead to an over-proportional inhibition of sucrose synthesis, the accumulation of phosphorylated intermediates and Pi-limitation of photosynthesis [24–26]. This inhibition occurs within minutes of the growth temperature falling a few degrees.
Cold acclimation includes a selective stimulation of sucrose synthesis and re-establishment of high rates of photosynthesis Recent studies with Arabidopsis have shown that a sequence of events reverses the inhibition of sucrose synthesis and photosynthesis as the plants acclimate to low temperatures [3,4,5••,16]. Short- and mid-term adjustments act primarily on sucrose synthesis but also stimulate photosynthesis by relieving the acute Pi-limitation. Longer-term adjustments affect photosynthesis directly. The recovery has two important functions: increased sucrose production [4] and protection against photoinhibition by allowing increased turnover of the photosynthetic electron chain [5••,9,27]. The transfer of warm-grown Arabidopsis plants to 4°C leads to the post-translational activation of SPS within 30 min. This is detected as a small shift of the apparent molecular weight and an increase in the substrate affinity of SPS. Inhibitor experiments indicate that SPS activation is due to phosphorylation, but the site has not yet been identified (E Hentschel, V Hurry, M Stitt, unpublished data). Over the next few days, sucrose synthesis is stimulated by two further adjustments. One is a selective increase in the expression of cFBP and SPS, the two key regulated enzymes in the pathway of sucrose synthesis [3,4,5••]. This leads to an increase in the levels of the transcripts and the encoded cFBP and SPS proteins. It also leads to a two- to three-fold increase in the activities of these enzymes relative to total leaf protein or relative to the activity of enzymes in the Calvin cycle and starch synthesis pathway. The second is a shift in the subcellular distribution of Pi. In the leaves of well-fertilised plants, most of the Pi is in the vacuole [28,29]. Indirect
evidence indicates that the Pi distribution shifts towards the cytoplasm at low temperatures [4,5••], allowing phosphorylated metabolites to increase without depleting the free Pi. The combination of increased substrate affinity, higher overall enzyme activities, and increased substrate levels compensates for the direct effects of low temperatures in reducing the rate of catalysis. Full acclimation occurs in leaves that develop at low temperature. They retain the selective increase in the expression of sucrose synthesis enzymes, and also have higher activities of all of the Calvin cycle enzymes on a fresh weight or leaf area basis [4,5••]. Two factors contribute to this increase. First, whereas transcripts for genes such as CHLOROPHYLL A/B-BINDING PROTEIN (CAB) and RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RBCS) decrease after transfer to low temperature, they recover in leaves that develop at low temperature [3]. This recovery occurs even though leaf sugars rise, indicating that sugar-repression of these genes [30] is overridden at low temperature in acclimatised leaves. Second, leaves that mature at low temperature have reduced water and increased protein contents compared to leaves that have developed at warmer temperatures [4,5••]. This is due to an increase in the volume of the cytoplasm relative to that of the vacuole [4,16]. Although studies with other species are less comprehensive, they indicate a similar picture to that seen in Arabidopsis. Post-translational activation of SPS occurs when potato tubers [31–33], apple fruits [34] or cabbage seedlings [7] are transferred to low temperature. The maximum activity of enzymes in the sucrose synthesis pathway increases during the cold acclimation of leaves from spinach [6], winter wheat, rye and rape [8,9], and of walnut wood [13]. Indirect evidence indicates that prolonged exposure to decreased temperatures leads to the gradual movement of Pi from the vacuole to the cytosol in spinach and barley leaves [29,35]. Cold-acclimated winter rye leaves [36,37] and wood parenchyma [38,39] have lower water contents but higher protein contents than the same tissues of plants that have not been chilled.
Acclimation of photosynthetic metabolism is triggered in part by acute Pi-limitation As already discussed, decreased temperatures lead to an acute Pi-limitation of photosynthesis. Intriguingly, some of the changes in photosynthetic metabolism that occur during cold acclimation are reminiscent of the response to low Pi [23,40,41]. Evidence that changes in Pi concentration or availability to metabolism contribute to cold acclimation has been provided by studies [5••] of pho1 [42] and pho2 [43] mutants. These mutants have decreased and increased shoot Pi concentrations, respectively. The low-temperatureinduced increase of SPS activity and of cFBP and SPS expression were accentuated in pho1 and attenuated in pho2. The decrease in the transcript levels of RBCS and CAB after chilling was abolished in pho1 and accentuated in
Photosynthetic carbon metabolism during cold acclimation Stitt and Hurry
pho2, and the cold-induced shift in carbon allocation from starch to sucrose was accentuated in pho1 and attenuated in pho2 [5••]. Proline accumulation after chilling was also decreased in pho2 and increased in pho1 when compared with wildtype plants. These results reveal that signals relating to altered Pi concentration or availability to metabolism lead to the activation and increased expression of enzymes in the sucrose synthesis pathway. They also lead to changes in the relative activities of enzymes in the Calvin cycle (see [5••] for details). Other components of the acclimation response, including the increase of leaf protein, were not modified in the pho1 and pho2 mutants [5••,16], revealing that they are not related to changes in Pi level. It is not known how the relative size of the vacuole and cytoplasm is regulated in plant cells, even though this is a crucial factor for plant performance. At normal temperatures, an increase in leaf protein concentration would be counterproductive because leaf area, and hence light absorption, would decrease. In addition, the increased rates of CO2 assimilation per unit area that are made possible by the higher protein concentration would not be matched by an increase in the rate of CO2 uptake (see [23] and references therein). The optimal leaf protein concentration will be higher at low temperatures because biochemical processes such as photosynthesis slow down far more as temperatures fall than do physical processes such as light absorption and CO2 entry. One speculative possibility is that the increased protein content at low temperatures is triggered by changes in the internal CO2 or O2 levels. Alternatively, low temperatures may interfere with leaf expansion, leading to a serendipitous increase of the protein concentration.
Further components of the cold-acclimation response include novel cold-induced polypeptides, changes in membrane fluidity and proline accumulation Low temperatures induce many other biochemical changes in addition to increased sugar levels. These include the accumulation of proline and, in some species, of other cryoprotectants including glycylbetaine [17,18••,44]; increased levels of antioxidants [10•]; and increased lipid desaturation to restore membrane fluidity [45,46]. Chilling also induces the expression of a number of COLD REGULATED (COR) genes [47,48,49•]. COR15a encodes a protein that interacts with lipids of the chloroplast envelope to maintain membrane function during freeze/thaw cycles [50]. Several other COR genes encode late-embyogenesislike proteins, indicating a function in desiccation tolerance, although their precise function is unknown [51]. In some cases, manipulating individual components alters cold tolerance. For example, transformants with increased lipid saturation are compromised in their ability to grow at low temperature [45], and transformants with increased levels of proline [52] or other cryoprotectants [44] show enhanced freezing tolerance. A major improvement in cold
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tolerance such as that seen in cold-adapted species may, however, require concerted changes of many components. Although the overexpression of individual COR genes does influence chloroplast freezing tolerance [51] or protoplast freezing tolerance [50], this strategy has not yet led to a significant improvement of freezing tolerance in whole plants. In contrast, overexpression of CBF1 [53-55] or CBF3 [18••] in Arabidopsis, or expression of CBF1, CBF2 or CBF3 in rape [56••], to drive a coordinated increase in the expression of a battery of COR genes led to a major improvement in frost tolerance.
Carbon metabolism interacts with other processes during cold acclimation In Synechocystis, two histidine kinases and a regulator response element are required for the induction at low temperatures of desB [57], a fatty acid desaturase implicated in increasing membrane fluidity at low temperatures. This pathway acts via additional uncharacterised response receptors to regulate the expression of many other genes, and parallel receptors may act on further groups of targets [57,58]. In higher plants, the primary temperature-sensing mechanism(s) have not yet been identified. However, the mechanism in plants is probably more complex than that in cyanobacteria because higher plants contain a large number of cells types, each with numerous subcellular compartments and membranes that differ in their function, composition and site(s) of synthesis. Molecular and genetic approaches have, however, uncovered a set of overlapping downstream pathways that are involved in cold-temperature signalling in Arabidopsis [10•,59•,60,61•]. Figure 1 summarises these pathways, and provides a tentative overview of their interactions with photosynthetic carbon metabolism. One well-characterized pathway involves the rapid induction of a small family of CBF/DREB1 transcription factors [47,48], which induce COR genes by binding at cold-responsive CRT/DRE elements in their promoters. The increase in COR transcript levels at low temperatures is independent of Pi [5••] and leaf sugar [15,17] concentrations. Overexpression of CBF leads to an increase in proline level, which is mediated via the induction of P5CS [18••], the first enzyme in the proline biosynthetic pathway. CBF overexpression also leads to an increase in sucrose synthesis. This must involve a less direct mechanism than that regulating proline accumulation, because SPS expression is unaffected [18••]. In a recent Arabidopsis microarray study [49•], several new cold-responsive genes were identified that contain a CRT/DRE element in their promoter region. One of these encoded enolase, a basic enzyme in central metabolism. The expression of individual COR genes may be a composite response to signals from multiple independent pathways. The CBF/DREB1 pathway is complemented by an abscisic acid (ABA)-dependent pathway that activates both a subset of the COR genes and a further sub-set of targets [60].
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Figure 1 ? ?
Sucrose Post-translational regulation of SPS Increased expression Cytoplasmic of SPS and cFBP Pi Re-distribution between Pi pools Photosynthetic gene expression Photosynthesis
Kinase/ Phosphatase
Ca2+ influx ?
esk1 sfr4 ?
Low temperature
Increased leaf protein
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Gene expression
? ABA
Chloroplast redox status
DREB1/CBF
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bZIP
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Current Opinion in Plant Biology
Signals involved in the cold acclimation of photosynthetic metabolism, and their interaction with other pathways involved in cold acclimation.
Of 19 cold-induced genes revealed in Arabidopsis microarrays [49•], half of the genes with CRT/DRE elements also contained an abscisic-acid-response element (ABRE) in their promoter. Although the post-translational activation of SPS is partly compromised in the ABA-deficient aba1-1 mutant, the gradual increase of total SPS activity and the recovery of photosynthesis after transfer to low temperature is unaffected in this mutant (Å Strand, M Stitt, unpublished data). This indicates that ABA-related signalling pathways do not play a major role in the cold acclimation of photosynthetic metabolism. Eskimo1 [17] and sfr4 [19,62] mutants are dramatically altered with respect to their ability to cold-acclimate, but their COR genes are induced by cold in the same way as those of wildtype plants. These mutations may define further low temperature signalling pathways or processes (see [19]), or loops that modulate the CBF/DREB1 pathway downstream of COR transcription. Sugar profiles are modified in eskimo1 and various sfr mutants [17,19,20], indicating that the pathways or loops defined by these mutations act directly or indirectly to alter sucrose concentrations. Work in cereals has revealed that the light environment or redox state of the chloroplast electron transport chain can induce the expression of chloroplast-localised cold-induced proteins [63,64••]. It is not yet known, however, whether sub-sets of the Arabidopsis COR genes are modulated in an analogous way. Light does lead to post-translational activation of SPS via a mechanism that interacts closely with changing Pi concentrations [65]. Low temperatures lead to a rapid increase in cytoplasmic Ca2+ concentration (see [10•,66] and references therein), and to a rapid Ca2+-dependent increase in protein phosphatase IIa (PPIIa) activity [67]. This may represent an early
step in some of the signalling pathways discussed above. Intriguingly, biochemical evidence implicates members of this group of protein phosphatases, as well as Ca2+-dependent kinases, in the post-translational regulation of enzymes of central metabolism, including SPS [62].
Possible interactions with vernalisation Studies with cereals have recently drawn attention to a further fascinating facet of cold acclimation. A set of COR genes have been identified in barley and winter wheat [68,69,70••]. In these species, prolonged exposure to low temperatures leads to a gradual decrease in the levels of COR transcripts and proteins, which is accompanied by a loss of cold tolerance [69,70••]. This loss of cold tolerance may be linked to the switch from the vegetative to the reproductive state as a result of vernalisation. Its dynamics depend on the vernalisation requirements of the cultivar, and are modified by day-length treatments that accelerate or delay the transition to reproductive growth [69,70••]. It will be interesting to learn whether similar interactions between vernalisation and cold tolerance occur in other species, including Arabidopsis in which mutants are available that are defective in the various signaling pathways for flower induction. These mutants would allow the mechanisms underlying the interaction to be dissected.
The physiological contribution of sugars in cold acclimation Sugars could act in several ways to promote cold acclimation (Figure 2) with most hypothetical mechanisms involving interactions with other components of the acclimation response. Dissection of the role of sugars in cold acclimation is a major task for the future. It has been proposed that sugars either act as osmotica or protect specific macromolecules during dehydration [2,71].
Photosynthetic carbon metabolism during cold acclimation Stitt and Hurry
Changes in the subcellular concentration and distribution of sugars might also provide a mechanism to protect specific compartments, or to regulate their volumes during cell expansion or dehydration. For example, although sucrose is largely restricted to the cytosol of leaves at high temperatures [28], there are reports that it accumulates in the chloroplast in cold-acclimated cabbage [72]. Decoding of the Arabidopsis genome has uncovered genes that could be involved in the production of specialised sugars that might be important in osmoregulation or cryoprotection, such as trehalose, raffinose and sugar alcohols [73••]. Tools that are becoming available in Arabidopsis will allow researchers to manipulate the cellular and subcellular levels, and the distribution, of specific sugars. Determining whether these modifications affect acclimation will make it possible to test critically the roles of specific sugars in osmoregulation and cryoprotection. Nothing is known about the interactions between COR gene products and sugars. If sugars contribute to the stabilisation of membrane structures, osmoregulation or subcellular volumes, they may act synergistically with or even as alternatives to some of the COR gene products. It is interesting that COR transcripts rise rapidly after transfer to the cold but decrease markedly in fully acclimated Arabidopsis plants [5••]. Changes in membrane fluidity will affect many aspects of metabolism at low temperature. Genetic lesions affecting fatty-acid elongation or desaturation have little effect on photosynthesis immediately after a fall in temperature, but lead over several cold days to ultrastructural changes, loss of chlorophyll and changes in chlorophyll fluorescence that indicate the occurrence of photoinhibition and the disruption of repair and assembly processes [45,74•]. This is consistent with the finding (see above) that low temperatures inhibit the ‘dark’ reactions of photosynthesis more strongly than they inhibit electron transport. Changes in membrane fluidity will affect metabolic processes in addition to photosynthesis. Mitochondria from the fad2 mutant have altered microviscosity, protein/lipid ratios, lipid mobility and proton leakage [75•]. An inappropriate membrane environment may affect transport proteins, which often exert significant control on metabolic fluxes [76]. It is well known that low temperatures inhibit phloem transport [2], though little is known about the underlying mechanism(s). The inhibition of growth at decreased temperatures in desA or desB mutants of Synechocystis correlates with the inhibition of their nitrate uptake [77], and is relieved by urea [78]. A concerted research effort is required to develop systems in which to assay membrane processes such as transport across membranes, vesicle dynamics, organelle biogenesis, and cell division and expansion. Understanding of these processes is necessary to understand how membrane fluidity affects metabolic and cellular processes, and to integrate studies of membrane fluidity with system-orientated analyses of metabolism and growth.
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Figure 2
Low temperature acclimation
Increased sucrose
Cryoprotection Osmoregulation
COR gene expression
Fatty acid desaturation
Proline synthesis
Current Opinion in Plant Biology
Possible roles of elevated sugar levels during cold acclimation and in frost tolerance. In addition to postulated roles as cryoprotectants and osmotica, sugars may be required as substrates and for the modulation of other energy- or assimilate-requiring processes during acclimation.
Sugars probably also have a more general and indirect role. It is intuitive that, at some point, falling sugar levels will interfere with other aspects of metabolism and cellular function, including responses to environmental challenges. Genetic analyses of sugar-sensing have concentrated on the response to high sugar concentrations [79,80]. The response to low sugar is likely to be at least as important [30]. For example, in tobacco, falling sugar concentrations lead to a dramatic inhibition of nitrogen assimilation and secondary metabolism [81,82]. This occurs via mechanisms that do not primarily involve decreased transcription (P Matt, M Stitt unpublished data). Although low sugars do not prevent the low-temperature-induced increase of COR transcripts, they do interfere with the development of frost tolerance [5••,15–17]. This raises the possibility that low sugars interfere with the synthesis, post-translational regulation or operation of COR proteins. It will be instructive to determine whether the constitutive frost tolerance provided by CBF overexpression is undermined by treatments that decrease sugar concentrations. Proline accumulation and fatty-acid turnover after transfer to low temperature are processes can be monitored to provide information about whether regulatory signals emanating from primary metabolism constrain or modulate cold acclimation. In this light, the acclimation of carbon metabolism may be an essential prerequisite for the adaptation and performance of plants at low temperatures.
Conclusions: wheels and roundabouts Even though overexpression of CBF family members leads to enhanced frost tolerance, the transformants grow poorly at normal temperatures [54,55]. This implies that at least some of the adjustments that occur during cold acclimation are detrimental at higher temperatures. The reasons for this will become clearer as more is learnt about the biochemical and cellular changes that occur during cold acclimation.
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Photosynthetic metabolism provides an easily approachable system to investigate the benefits and cost of cold adaptation. On the basis of previous research (see above), specific predictions can be made about why increased expression of the sucrose synthesis pathway or an increased leaf protein concentration might be disadvantageous at higher temperatures, and methods are available to test these predictions by investigating gas exchange and metabolite levels.
as a signals in the regulation of photosynthetic/metabolic acclimation to low temperatures, and lead to major changes in the ability of the different genotypes to develop frost tolerance.
In conclusion, cold acclimation is a multi-facetted response to a pervasive problem. In the era of functional genomics and system-orientated research, it provides a fascinating test case to explore interactions between areas of plant function that have been investigated in isolation in the past. By investigating cold acclimation, researchers can begin to understand how events in primary metabolism contribute to, influence and constrain changes in biosynthetic metabolism, cellular function and development. The information generated by focused studies of gene function in Arabidopsis will be the springboard for a new wave of strategies to improve cold- and frost-tolerance in crop plants.
Acknowledgements The authors’ work was supported by the Alexander von Humboldt Society, the Deutsche Forschungsgemeinschaft, the GABI programme of the German Ministry for Education Research, and the Swedish Council for Forestry and Agricultural Research. We are grateful to Åsa Strand, Eike Hentschel and Per Gardeström for cooperation. This article is dedicated to the memory of Peter Steponkus.
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49. Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K, • Carninci P, Hayashizaki Y, Shinozaki K: Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell 2001, 13:61-72. The first published transcript-array study in higher plants to be carried out at low temperatures. The authors identify a number of cold-induced genes for the first time, some with CRT/DRE elements in their promoters. About half of these also contain ABRE elements in their promoters, demonstrating the broad crossover between these signalling pathways. The authors also identify enolase as a cold-induced protein that, intriguingly, contains a CRT/DRE element in its promoter region.
30. Koch KE: Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 1996, 47:509-540. 31. Hill LM, Reimholz R, Schroder R, Nielsen TH, Stitt M: The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase. Plant Cell Environ 1996, 19:1223-1237. 32. Deiting U, Zrenner R, Stitt M: Similar temperature requirements for sugar accumulation and for the induction of new forms of sucrose phosphate synthase and amylase in cold-stored potato tubers. Plant Cell Environ 1998, 21:127-138.
50. Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF: Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 1998, 95:14570-14575. 51. Thomashow MF: Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 1998, 118:1-7.
33. Krause KP, Hill L, Reimholz R, Nielsen TH, Sonnewald U, Stitt M: Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase. Plant Cell Environ 1998, 21:285-299.
52. Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K: Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 1999, 461:205-210.
34. Duque P, Barreiro MG, Arrabaca JD: Respiratory metabolism during cold storage of apple fruit. I. Sucrose metabolism and glycolysis. Physiol Plant 1999, 107:14-23.
53. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF: Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 1998, 280:104-106.
35. Labate CA, Leegood RC: Limitation of photosynthesis by changes in temperature. Factors affecting the response of carbon dioxide assimilation to temperature in barley leaves. Planta 1988, 173:519-527. 36. Huner NPA, Palta JP, Li PH, Carter JV: Anatomical changes in leaves of Puma rye in response to growth at cold-hardening temperatures. Bot Gaz 1981, 142:55-62. 37.
Griffith M, Huner NPA, Espelie KE, Kolattukudy PE: Lipid polymers accumulate in the epidermis and mestome sheath cell walls during low temperature development of winter rye leaves. Protoplasma 1985, 125:53-64.
38. Siminovitch D, Rheume B, Pomeroy K, Lepage M: Phospholipid, protein, and nucleic acid increases in protoplasm and membrane structures associated with development of extreme freezing resistance in black locust tree cells. Cryobiology 1968, 5:202-225. 39. Sauter JJ, Wisniewski M, Witt W: Interrelationships between ultrastructure, sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation and deacclimation in poplar (Populus x canadensis Moench [robusta]) wood. J Plant Physiol 1996, 149:451-461. 40. Taiz L, Zeiger E: Plant Physiology edn 2. Sunderland, Massachusetts: Sinauer Associates Inc; 1998. 41. Nielsen TH, Krapp A, Roper-Schwarz U, Stitt M: The sugar-mediated regulation of genes encoding the small subunit of Rubisco and the regulatory subunit of ADP glucose pyrophosphorylase is modified by phosphate and nitrogen. Plant Cell Environ 1998, 21:443-454. 42. Poirier Y, Thoma S, Somerville C, Schiefelbein J: A mutant of Arabidopsis deficient in xylem loading of phosphate. Plant Physiol 1991, 97:1087-1093. 43. Delhaize E, Randall PJ: Characterization of a phosphateaccumulator mutant of Arabidopsis thaliana. Plant Physiol 1995, 107:207-213.
54. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K: Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperatureresponsive gene expression, respectively, in Arabidopsis. Plant Cell 1998, 10:1391-1406. 55. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K: Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 1999, 17:287-291. 56. Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, •• Thomashow MF: Components of the Arabidopsis Crepeat/dehydration-responsive element binding factor coldresponse pathway are conserved in Brassica napus and other plant species. Plant Physiol 2001, 127:910-917. An exemplary use of information gained in the model species Arabidopsis to improve traits in crop plants. The cold-induced transcription factors CBF1, CBF2 and CBF3 from Arabidopsis were overexpressed in Brassica napus and shown to drive COR gene expression and improve frost hardiness. 57.
Suzuki I, Los DA, Kanesaki Y, Mikami K, Murata N: The pathway for perception and transduction of low-temperature signals in Synechocystis. EMBO J 2000, 19:1327-1334.
58. Suzuki I, Kanesaki Y, Mikami K, Kanehisa M, Murata N: Coldregulated genes under control of the cold sensor Hik33 in Synechocystis. Mol Microbiol 2001, 40:235-244. 59. Thomashow MF: So what’s new in the field of plant cold • acclimation? Lots! Plant Physiol 2001, 125:89-93. A good recent review of progress in the field of plant cold acclimation. 60. Shinozaki K, Yamaguchi-Shinozaki K: Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 2000, 3:217-223.
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61. Browse J, Xin ZG: Temperature sensing and cold acclimation. Curr • Opin Plant Biol 2001, 4:241-246. A good review in which the authors discuss how multiple signalling pathways and probably multiple temperature sensors are used to initiate and control cold acclimation in higher plants. 62. Knight H, Veale EL, Warren GJ, Knight MR: The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 1999, 11:875-886. 63. Gray GR, Chauvin L-P, Sarhan F, Huner NPA: Cold acclimation and freezing tolerance: a complex interaction of light and temperature. Plant Physiol 1997, 114:467-474. 64. Ndong C, Danyluk J, Huner NPA, Sarhan F: Survey of gene •• expression in winter rye during changes in growth temperature, irradiance or excitation pressure. Plant Mol Biol 2001, 45:691-703. Expression of a chloroplast-targeted COR gene in cereals is responsive not only to low temperature but also to intense light, indicating that the expression of this gene may be controlled by a signal originating in the chloroplast, as well as by signal(s) originating at the plasma membrane. 65. Toroser D, Huber SC: Carbon and nitrogen metabolism and reversible protein phosphorylation. Adv Bot Res 2000, 32:435-458. 66. Knight H, Brandt S, Knight MR: A history of stress alters drought calcium signalling pathways in Arabidopsis. Plant J 1998, 16:681-687. 67.
Monroy AF, Sangwan V, Dhindsa RS: Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold-inactivation. Plant J 1998, 13:653-660.
68. Oliver AE, Crowe LM, Crowe JH: Methods for dehydrationtolerance: depression of the phase transition temperature in dry membranes and carbohydrate vitrification. Seed Sci Res 1998, 8:211-221. 69. Santarius KA, Milde H: Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells. Planta 1977, 136:163-166. 70. The Arabidopsis Genome Initiative: Analysis of the genome •• sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408:796-815. A treasure trove of information that, in combination with developing genetic resources, is revolutionising plant physiology. The work described in this paper provides the enabling step for a systematic investigation of gene function and its relation to plant phenotype. This will be the paradigm for investigations of cold acclimation in the future and will form the foundation for major breakthroughs in breeding crops to cope with marginal and adverse temperatures. On a more specific note, analysis of the Arabidopsis genome sequence indicates the presence of genes that synthesise a range of unusual sugars, which could be involved in specialised functions. 71. Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F: Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 1998, 10:623-638. 72. Fowler DB, Breton G, Limin AE, Mahfoozi S, Sarhan F: Photoperiod and temperature interactions regulate low-temperature-induced gene expression in barley. Plant Physiol 2001, 127:1676-1681.
73. Mahfoozi S, Limin AE, Fowler DB: Developmental regulation of •• low-temperature tolerance in winter wheat. Ann Bot 2001, 87:751-757. The authors had previously presented evidence that, in barley [72] and wheat [71], COR gene expression and protein accumulation is controlled in the longer term by the vernalisation requirement and the photoperiod response. Low-temperature tolerance and the expression of COR genes in cereals are greatest in plants that are in the vegetative state. Both decline with time at low temperature as vernalisation leads to the switch from vegetative to reproductive growth. The decline in low-temperature tolerance and the expression of COR genes can be accelerated by long days, which also promote the switch from vegetative to reproductive growth. The authors propose that the expression of the COR genes in cereals, and the attainment and maintenance of low temperature tolerance, is related to the stage of phonological development. 74. Routaboul JM, Fischer SF, Browse J: Trienoic fatty acids are • required to maintain chloroplast function at low temperatures. Plant Physiol 2000, 124:1697-1705. A particularly thorough investigation of the response of photosynthesis to the loss of unsaturated fatty acids. This work provides further evidence that inappropriate saturation does not seriously impair the short-term response of photosynthesis, but instead leads over a number of days to deficiencies in chloroplast function. 75. Caiveau O, Fortune D, Cantrel C, Zachowski A, Moreau F: • Consequences of omega-6-oleate desaturase deficiency on lipid dynamics and functional properties of mitochondrial membranes of Arabidopsis thaliana. J Biol Chem 2001, 276:5788-5794. Most biochemical research on the effects of inappropriate fatty acid saturation in planta has concentrated on thylakoid function. Nevertheless, there is substantial evidence that other metabolic and physiological functions are affected more severely. The authors present a detailed analysis of the effect of increased lipid saturation on mitochondrial structure and on coupling in isolated mitochondria. They show that increased lipid saturation leads to changes in mitochondrial structure, and impairs coupling in isolated mitochondria. 76. Stitt M, Sonnewald U: Regulation of metabolism in transgenic plants. Annu Rev Plant Physiol Plant Mol Biol 1995, 46:341-368. 77.
Sakamoto T, Bryant DA: Nitrate transport and not photoinhibition limits growth of the freshwater cyanobacterium Synechococcus species PCC 6301 at low temperature. Plant Physiol 1999, 119:785-794.
78. Sakamoto T, Shen GZ, Higashi S, Murata N, Bryant DA: Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation. Arch Microbiol 1998, 169:20-28. 79. Sheen J, Zhou L, Jang JC: Sugars as signaling molecules. Curr Opin Plant Biol 1999, 2:410-418. 80. Smeekens S: Sugar-induced signal transduction in plants. Annu Rev Plant Physiol Plant Mol Biol 2000, 51:49-81. 81. Matt P, Schurr U, Klein D, Krapp A, Stitt M: Growth of tobacco in shortday conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis. Planta 1998, 207:27-41. 82. Matt P, Krapp A, Haake V, Mock HP, Stitt M: Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants. Plant J 2002, in press.
A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis Mark Stitt* and Vaughan Hurry† Low temperatures lead to the inhibition of sucrose synthesis and photosynthesis. The biochemical and physiological adaptations of plants to low temperatures include the posttranslational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in the leaf protein content. Recent progress has been made in understanding both the signals that trigger these processes and how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation. Addresses *Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany; e-mail: [email protected] †Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Sweden Correspondence: Mark Stitt Current Opinion in Plant Biology 2002, 5:199–206 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 21 March 2002 Abbreviations ABA abscisic acid CBF CRT-binding factor cFBPase cytosolic fructose-1,6-bisphosphatase COR COLD REGULATED CRT C-repeat desB fatty acid desaturaseB DRE dehydration-responsive element DREB1 DRE-binding protein1 Pi inorganic orthophosphate Ru1,5bisP ribulose-1,5-bisphosphate sfr4 sensitive-to-freezing4 SPS sucrose phosphate synthase
Introduction Low temperature is one of the most important factors affecting plant performance and distribution [1,2]. At high latitudes or altitudes, the problem of coping with low temperatures is exacerbated by the need to prolong the growing season beyond the short summer. Low temperatures slow down enzyme-catalysed reactions and modify the conformation of lipids and other macromolecules, with consequences for most biological processes. It is difficult to determine which processes are affected most severely by cold, and differential responses generate complex indirect effects. Sub-freezing temperatures lead to severe damage caused by dehydration when ice forms outside the cell or, in extreme cases, to freezing of the cytoplasm. Many species, including crops such as maize, bean, tomato and potato, have only a limited capacity to cope with low
temperatures [2]. Cold-hardy herbaceous species, including Arabidopsis [3,4,5••] and crops such as winter cereals, winter rape, spinach and cabbage [6–9], grow at low temperatures and survive freezing temperatures. Biennials and woody perennials that are adapted to high latitudes cope with temperatures down to –40°C or lower [2]. Low temperature tolerance develops in cold-hardy species during a period of exposure to low but non-freezing temperatures in a multifacetted process termed cold-acclimation [10•]. In this review, we discuss the biochemical and physiological adaptations of plants to low temperature. These include the post-translational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in leaf protein content. We then discuss signals that may trigger these processes and consider how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation.
An important role for sugars in cold acclimation Descriptive ecological and agronomic studies have uncovered a strong correlation between sugar concentrations and frost resistance [2,7,11–14]. Likewise, changes of light regime during cold acclimation revealed a strong correlation between sugar levels and frost tolerance in Arabidopsis [15]. The sugar concentrations in transformants with antisense inhibition of cytosolic fructose-1,6-bisphosphatase (cFBPase) and sucrose phosphate synthase (SPS) expression, or with overexpression of maize SPS, correlated with the extent of frost tolerance after cold acclimation [16]. Similarly, the constitutive increases in the frost tolerance of the eskimo1 mutant [17] and in transformants over-expressing the C-repeat (CRT)-binding factor (CBF)/dehydration-responsive element (DRE)-binding protein1 (DREB1) [18••] correlated with increased sugar contents. The sensitive-to-freezing4 (sfr4) mutant is impaired in cold acclimation and has sugar levels that are lower than those of wildtype Arabidopsis plants [19,20]. Recent research has started to define the functional importance of sugar metabolism during cold acclimation. Progress has been achieved by investigating which adaptations in photosynthetic carbon metabolism provide increased sugar levels, which signals are involved, and how sugar metabolism interacts with other responses during cold acclimation.
An over-proportional inhibition of sucrose synthesis reduces photosynthesis at low temperatures During photosynthesis, CO2 combines with ribulose-1,5bisphosphate (Ru1,5bisP) to form glycerate-3-phosphate, which is reduced to triose-phosphate using NADPH and
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ATP that is generated by photosynthetic electron transport. The majority of the triose phosphates are retained in the chloroplast to regenerate Ru1,5bisP. The surplus is converted to end products, releasing inorganic orthophosphate (Pi) that recombines with ADP to regenerate ATP. The most important pathway for end-product synthesis involves the export of triose phosphates to the cytosol, where they are converted to sucrose via cFBPase and SPS. Optimal rates of photosynthesis require an appropriate balance between the rates of carbon fixation and sucrose synthesis [21–23]. Excessive sucrose synthesis depletes the phosphorylated Calvin cycle intermediates and inhibits the regeneration of Ru1,5bisP. Conversely, inadequate sucrose synthesis leads to accumulation of phosphorylated intermediates and depletion of Pi, resulting in inhibition of ATP synthesis, accumulation of glycerate3-phosphate and inactivation of Rubisco. Studies performed in the late 1980s with barley and spinach showed that falling temperatures lead to an over-proportional inhibition of sucrose synthesis, the accumulation of phosphorylated intermediates and Pi-limitation of photosynthesis [24–26]. This inhibition occurs within minutes of the growth temperature falling a few degrees.
Cold acclimation includes a selective stimulation of sucrose synthesis and re-establishment of high rates of photosynthesis Recent studies with Arabidopsis have shown that a sequence of events reverses the inhibition of sucrose synthesis and photosynthesis as the plants acclimate to low temperatures [3,4,5••,16]. Short- and mid-term adjustments act primarily on sucrose synthesis but also stimulate photosynthesis by relieving the acute Pi-limitation. Longer-term adjustments affect photosynthesis directly. The recovery has two important functions: increased sucrose production [4] and protection against photoinhibition by allowing increased turnover of the photosynthetic electron chain [5••,9,27]. The transfer of warm-grown Arabidopsis plants to 4°C leads to the post-translational activation of SPS within 30 min. This is detected as a small shift of the apparent molecular weight and an increase in the substrate affinity of SPS. Inhibitor experiments indicate that SPS activation is due to phosphorylation, but the site has not yet been identified (E Hentschel, V Hurry, M Stitt, unpublished data). Over the next few days, sucrose synthesis is stimulated by two further adjustments. One is a selective increase in the expression of cFBP and SPS, the two key regulated enzymes in the pathway of sucrose synthesis [3,4,5••]. This leads to an increase in the levels of the transcripts and the encoded cFBP and SPS proteins. It also leads to a two- to three-fold increase in the activities of these enzymes relative to total leaf protein or relative to the activity of enzymes in the Calvin cycle and starch synthesis pathway. The second is a shift in the subcellular distribution of Pi. In the leaves of well-fertilised plants, most of the Pi is in the vacuole [28,29]. Indirect
evidence indicates that the Pi distribution shifts towards the cytoplasm at low temperatures [4,5••], allowing phosphorylated metabolites to increase without depleting the free Pi. The combination of increased substrate affinity, higher overall enzyme activities, and increased substrate levels compensates for the direct effects of low temperatures in reducing the rate of catalysis. Full acclimation occurs in leaves that develop at low temperature. They retain the selective increase in the expression of sucrose synthesis enzymes, and also have higher activities of all of the Calvin cycle enzymes on a fresh weight or leaf area basis [4,5••]. Two factors contribute to this increase. First, whereas transcripts for genes such as CHLOROPHYLL A/B-BINDING PROTEIN (CAB) and RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RBCS) decrease after transfer to low temperature, they recover in leaves that develop at low temperature [3]. This recovery occurs even though leaf sugars rise, indicating that sugar-repression of these genes [30] is overridden at low temperature in acclimatised leaves. Second, leaves that mature at low temperature have reduced water and increased protein contents compared to leaves that have developed at warmer temperatures [4,5••]. This is due to an increase in the volume of the cytoplasm relative to that of the vacuole [4,16]. Although studies with other species are less comprehensive, they indicate a similar picture to that seen in Arabidopsis. Post-translational activation of SPS occurs when potato tubers [31–33], apple fruits [34] or cabbage seedlings [7] are transferred to low temperature. The maximum activity of enzymes in the sucrose synthesis pathway increases during the cold acclimation of leaves from spinach [6], winter wheat, rye and rape [8,9], and of walnut wood [13]. Indirect evidence indicates that prolonged exposure to decreased temperatures leads to the gradual movement of Pi from the vacuole to the cytosol in spinach and barley leaves [29,35]. Cold-acclimated winter rye leaves [36,37] and wood parenchyma [38,39] have lower water contents but higher protein contents than the same tissues of plants that have not been chilled.
Acclimation of photosynthetic metabolism is triggered in part by acute Pi-limitation As already discussed, decreased temperatures lead to an acute Pi-limitation of photosynthesis. Intriguingly, some of the changes in photosynthetic metabolism that occur during cold acclimation are reminiscent of the response to low Pi [23,40,41]. Evidence that changes in Pi concentration or availability to metabolism contribute to cold acclimation has been provided by studies [5••] of pho1 [42] and pho2 [43] mutants. These mutants have decreased and increased shoot Pi concentrations, respectively. The low-temperatureinduced increase of SPS activity and of cFBP and SPS expression were accentuated in pho1 and attenuated in pho2. The decrease in the transcript levels of RBCS and CAB after chilling was abolished in pho1 and accentuated in
Photosynthetic carbon metabolism during cold acclimation Stitt and Hurry
pho2, and the cold-induced shift in carbon allocation from starch to sucrose was accentuated in pho1 and attenuated in pho2 [5••]. Proline accumulation after chilling was also decreased in pho2 and increased in pho1 when compared with wildtype plants. These results reveal that signals relating to altered Pi concentration or availability to metabolism lead to the activation and increased expression of enzymes in the sucrose synthesis pathway. They also lead to changes in the relative activities of enzymes in the Calvin cycle (see [5••] for details). Other components of the acclimation response, including the increase of leaf protein, were not modified in the pho1 and pho2 mutants [5••,16], revealing that they are not related to changes in Pi level. It is not known how the relative size of the vacuole and cytoplasm is regulated in plant cells, even though this is a crucial factor for plant performance. At normal temperatures, an increase in leaf protein concentration would be counterproductive because leaf area, and hence light absorption, would decrease. In addition, the increased rates of CO2 assimilation per unit area that are made possible by the higher protein concentration would not be matched by an increase in the rate of CO2 uptake (see [23] and references therein). The optimal leaf protein concentration will be higher at low temperatures because biochemical processes such as photosynthesis slow down far more as temperatures fall than do physical processes such as light absorption and CO2 entry. One speculative possibility is that the increased protein content at low temperatures is triggered by changes in the internal CO2 or O2 levels. Alternatively, low temperatures may interfere with leaf expansion, leading to a serendipitous increase of the protein concentration.
Further components of the cold-acclimation response include novel cold-induced polypeptides, changes in membrane fluidity and proline accumulation Low temperatures induce many other biochemical changes in addition to increased sugar levels. These include the accumulation of proline and, in some species, of other cryoprotectants including glycylbetaine [17,18••,44]; increased levels of antioxidants [10•]; and increased lipid desaturation to restore membrane fluidity [45,46]. Chilling also induces the expression of a number of COLD REGULATED (COR) genes [47,48,49•]. COR15a encodes a protein that interacts with lipids of the chloroplast envelope to maintain membrane function during freeze/thaw cycles [50]. Several other COR genes encode late-embyogenesislike proteins, indicating a function in desiccation tolerance, although their precise function is unknown [51]. In some cases, manipulating individual components alters cold tolerance. For example, transformants with increased lipid saturation are compromised in their ability to grow at low temperature [45], and transformants with increased levels of proline [52] or other cryoprotectants [44] show enhanced freezing tolerance. A major improvement in cold
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tolerance such as that seen in cold-adapted species may, however, require concerted changes of many components. Although the overexpression of individual COR genes does influence chloroplast freezing tolerance [51] or protoplast freezing tolerance [50], this strategy has not yet led to a significant improvement of freezing tolerance in whole plants. In contrast, overexpression of CBF1 [53-55] or CBF3 [18••] in Arabidopsis, or expression of CBF1, CBF2 or CBF3 in rape [56••], to drive a coordinated increase in the expression of a battery of COR genes led to a major improvement in frost tolerance.
Carbon metabolism interacts with other processes during cold acclimation In Synechocystis, two histidine kinases and a regulator response element are required for the induction at low temperatures of desB [57], a fatty acid desaturase implicated in increasing membrane fluidity at low temperatures. This pathway acts via additional uncharacterised response receptors to regulate the expression of many other genes, and parallel receptors may act on further groups of targets [57,58]. In higher plants, the primary temperature-sensing mechanism(s) have not yet been identified. However, the mechanism in plants is probably more complex than that in cyanobacteria because higher plants contain a large number of cells types, each with numerous subcellular compartments and membranes that differ in their function, composition and site(s) of synthesis. Molecular and genetic approaches have, however, uncovered a set of overlapping downstream pathways that are involved in cold-temperature signalling in Arabidopsis [10•,59•,60,61•]. Figure 1 summarises these pathways, and provides a tentative overview of their interactions with photosynthetic carbon metabolism. One well-characterized pathway involves the rapid induction of a small family of CBF/DREB1 transcription factors [47,48], which induce COR genes by binding at cold-responsive CRT/DRE elements in their promoters. The increase in COR transcript levels at low temperatures is independent of Pi [5••] and leaf sugar [15,17] concentrations. Overexpression of CBF leads to an increase in proline level, which is mediated via the induction of P5CS [18••], the first enzyme in the proline biosynthetic pathway. CBF overexpression also leads to an increase in sucrose synthesis. This must involve a less direct mechanism than that regulating proline accumulation, because SPS expression is unaffected [18••]. In a recent Arabidopsis microarray study [49•], several new cold-responsive genes were identified that contain a CRT/DRE element in their promoter region. One of these encoded enolase, a basic enzyme in central metabolism. The expression of individual COR genes may be a composite response to signals from multiple independent pathways. The CBF/DREB1 pathway is complemented by an abscisic acid (ABA)-dependent pathway that activates both a subset of the COR genes and a further sub-set of targets [60].
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Figure 1 ? ?
Sucrose Post-translational regulation of SPS Increased expression Cytoplasmic of SPS and cFBP Pi Re-distribution between Pi pools Photosynthetic gene expression Photosynthesis
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Signals involved in the cold acclimation of photosynthetic metabolism, and their interaction with other pathways involved in cold acclimation.
Of 19 cold-induced genes revealed in Arabidopsis microarrays [49•], half of the genes with CRT/DRE elements also contained an abscisic-acid-response element (ABRE) in their promoter. Although the post-translational activation of SPS is partly compromised in the ABA-deficient aba1-1 mutant, the gradual increase of total SPS activity and the recovery of photosynthesis after transfer to low temperature is unaffected in this mutant (Å Strand, M Stitt, unpublished data). This indicates that ABA-related signalling pathways do not play a major role in the cold acclimation of photosynthetic metabolism. Eskimo1 [17] and sfr4 [19,62] mutants are dramatically altered with respect to their ability to cold-acclimate, but their COR genes are induced by cold in the same way as those of wildtype plants. These mutations may define further low temperature signalling pathways or processes (see [19]), or loops that modulate the CBF/DREB1 pathway downstream of COR transcription. Sugar profiles are modified in eskimo1 and various sfr mutants [17,19,20], indicating that the pathways or loops defined by these mutations act directly or indirectly to alter sucrose concentrations. Work in cereals has revealed that the light environment or redox state of the chloroplast electron transport chain can induce the expression of chloroplast-localised cold-induced proteins [63,64••]. It is not yet known, however, whether sub-sets of the Arabidopsis COR genes are modulated in an analogous way. Light does lead to post-translational activation of SPS via a mechanism that interacts closely with changing Pi concentrations [65]. Low temperatures lead to a rapid increase in cytoplasmic Ca2+ concentration (see [10•,66] and references therein), and to a rapid Ca2+-dependent increase in protein phosphatase IIa (PPIIa) activity [67]. This may represent an early
step in some of the signalling pathways discussed above. Intriguingly, biochemical evidence implicates members of this group of protein phosphatases, as well as Ca2+-dependent kinases, in the post-translational regulation of enzymes of central metabolism, including SPS [62].
Possible interactions with vernalisation Studies with cereals have recently drawn attention to a further fascinating facet of cold acclimation. A set of COR genes have been identified in barley and winter wheat [68,69,70••]. In these species, prolonged exposure to low temperatures leads to a gradual decrease in the levels of COR transcripts and proteins, which is accompanied by a loss of cold tolerance [69,70••]. This loss of cold tolerance may be linked to the switch from the vegetative to the reproductive state as a result of vernalisation. Its dynamics depend on the vernalisation requirements of the cultivar, and are modified by day-length treatments that accelerate or delay the transition to reproductive growth [69,70••]. It will be interesting to learn whether similar interactions between vernalisation and cold tolerance occur in other species, including Arabidopsis in which mutants are available that are defective in the various signaling pathways for flower induction. These mutants would allow the mechanisms underlying the interaction to be dissected.
The physiological contribution of sugars in cold acclimation Sugars could act in several ways to promote cold acclimation (Figure 2) with most hypothetical mechanisms involving interactions with other components of the acclimation response. Dissection of the role of sugars in cold acclimation is a major task for the future. It has been proposed that sugars either act as osmotica or protect specific macromolecules during dehydration [2,71].
Photosynthetic carbon metabolism during cold acclimation Stitt and Hurry
Changes in the subcellular concentration and distribution of sugars might also provide a mechanism to protect specific compartments, or to regulate their volumes during cell expansion or dehydration. For example, although sucrose is largely restricted to the cytosol of leaves at high temperatures [28], there are reports that it accumulates in the chloroplast in cold-acclimated cabbage [72]. Decoding of the Arabidopsis genome has uncovered genes that could be involved in the production of specialised sugars that might be important in osmoregulation or cryoprotection, such as trehalose, raffinose and sugar alcohols [73••]. Tools that are becoming available in Arabidopsis will allow researchers to manipulate the cellular and subcellular levels, and the distribution, of specific sugars. Determining whether these modifications affect acclimation will make it possible to test critically the roles of specific sugars in osmoregulation and cryoprotection. Nothing is known about the interactions between COR gene products and sugars. If sugars contribute to the stabilisation of membrane structures, osmoregulation or subcellular volumes, they may act synergistically with or even as alternatives to some of the COR gene products. It is interesting that COR transcripts rise rapidly after transfer to the cold but decrease markedly in fully acclimated Arabidopsis plants [5••]. Changes in membrane fluidity will affect many aspects of metabolism at low temperature. Genetic lesions affecting fatty-acid elongation or desaturation have little effect on photosynthesis immediately after a fall in temperature, but lead over several cold days to ultrastructural changes, loss of chlorophyll and changes in chlorophyll fluorescence that indicate the occurrence of photoinhibition and the disruption of repair and assembly processes [45,74•]. This is consistent with the finding (see above) that low temperatures inhibit the ‘dark’ reactions of photosynthesis more strongly than they inhibit electron transport. Changes in membrane fluidity will affect metabolic processes in addition to photosynthesis. Mitochondria from the fad2 mutant have altered microviscosity, protein/lipid ratios, lipid mobility and proton leakage [75•]. An inappropriate membrane environment may affect transport proteins, which often exert significant control on metabolic fluxes [76]. It is well known that low temperatures inhibit phloem transport [2], though little is known about the underlying mechanism(s). The inhibition of growth at decreased temperatures in desA or desB mutants of Synechocystis correlates with the inhibition of their nitrate uptake [77], and is relieved by urea [78]. A concerted research effort is required to develop systems in which to assay membrane processes such as transport across membranes, vesicle dynamics, organelle biogenesis, and cell division and expansion. Understanding of these processes is necessary to understand how membrane fluidity affects metabolic and cellular processes, and to integrate studies of membrane fluidity with system-orientated analyses of metabolism and growth.
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Figure 2
Low temperature acclimation
Increased sucrose
Cryoprotection Osmoregulation
COR gene expression
Fatty acid desaturation
Proline synthesis
Current Opinion in Plant Biology
Possible roles of elevated sugar levels during cold acclimation and in frost tolerance. In addition to postulated roles as cryoprotectants and osmotica, sugars may be required as substrates and for the modulation of other energy- or assimilate-requiring processes during acclimation.
Sugars probably also have a more general and indirect role. It is intuitive that, at some point, falling sugar levels will interfere with other aspects of metabolism and cellular function, including responses to environmental challenges. Genetic analyses of sugar-sensing have concentrated on the response to high sugar concentrations [79,80]. The response to low sugar is likely to be at least as important [30]. For example, in tobacco, falling sugar concentrations lead to a dramatic inhibition of nitrogen assimilation and secondary metabolism [81,82]. This occurs via mechanisms that do not primarily involve decreased transcription (P Matt, M Stitt unpublished data). Although low sugars do not prevent the low-temperature-induced increase of COR transcripts, they do interfere with the development of frost tolerance [5••,15–17]. This raises the possibility that low sugars interfere with the synthesis, post-translational regulation or operation of COR proteins. It will be instructive to determine whether the constitutive frost tolerance provided by CBF overexpression is undermined by treatments that decrease sugar concentrations. Proline accumulation and fatty-acid turnover after transfer to low temperature are processes can be monitored to provide information about whether regulatory signals emanating from primary metabolism constrain or modulate cold acclimation. In this light, the acclimation of carbon metabolism may be an essential prerequisite for the adaptation and performance of plants at low temperatures.
Conclusions: wheels and roundabouts Even though overexpression of CBF family members leads to enhanced frost tolerance, the transformants grow poorly at normal temperatures [54,55]. This implies that at least some of the adjustments that occur during cold acclimation are detrimental at higher temperatures. The reasons for this will become clearer as more is learnt about the biochemical and cellular changes that occur during cold acclimation.
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Photosynthetic metabolism provides an easily approachable system to investigate the benefits and cost of cold adaptation. On the basis of previous research (see above), specific predictions can be made about why increased expression of the sucrose synthesis pathway or an increased leaf protein concentration might be disadvantageous at higher temperatures, and methods are available to test these predictions by investigating gas exchange and metabolite levels.
as a signals in the regulation of photosynthetic/metabolic acclimation to low temperatures, and lead to major changes in the ability of the different genotypes to develop frost tolerance.
In conclusion, cold acclimation is a multi-facetted response to a pervasive problem. In the era of functional genomics and system-orientated research, it provides a fascinating test case to explore interactions between areas of plant function that have been investigated in isolation in the past. By investigating cold acclimation, researchers can begin to understand how events in primary metabolism contribute to, influence and constrain changes in biosynthetic metabolism, cellular function and development. The information generated by focused studies of gene function in Arabidopsis will be the springboard for a new wave of strategies to improve cold- and frost-tolerance in crop plants.
Acknowledgements The authors’ work was supported by the Alexander von Humboldt Society, the Deutsche Forschungsgemeinschaft, the GABI programme of the German Ministry for Education Research, and the Swedish Council for Forestry and Agricultural Research. We are grateful to Åsa Strand, Eike Hentschel and Per Gardeström for cooperation. This article is dedicated to the memory of Peter Steponkus.
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