JXB Advance Access originally published online on April 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 387, pp. 1565-1575,
June 1, 2003
© 2003 Oxford University Press
Sugars regulate cold-induced gene expression and freezing-tolerance in barley cell cultures
Received 19 November 2002; Accepted 19 March 2003
School of Biology, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK
1 To whom correspondence should be addressed. Fax: +44 191 222 8684. E-mail: R.S.Pearce{at}ncl.ac.uk
2 Present address: Department of Genetics and Plant Physiology, Research Institute of Forests and Rangelands, PO Box 13185-116, Tehran, Iran.
Abbreviations: EF, elongation factor; nsLTP, non-specific lipid transfer protein; TTC, 2,3,5-triphenyl tetrazolium chloride.
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Abstract |
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The hypothesis that the extracellular concentration of sugars helps regulate the acclimation of plant cells to cold was tested in this work. Suspension cultures were used to control the concentration of sugars in the medium supplied to barley cell cultures (Hordeum vulgare L. cv. Igri), replacing the medium daily to help maintain the concentration. Freezing tolerance and the levels of mRNA expression of the stress-response genes blt4.9 (coding for a non- specific lipid transfer protein) and dhn1 (coding for a dehydrin) were measured. Similar levels of freezing-tolerance and gene expression were obtained in the experiments as occur during cold-acclimation in the crown of the whole plant. In the cell cultures, cold (6/2 °C) did not induce an increase in freezing tolerance or in the expression of detectable levels of blt4.9 or dhn1 mRNAs when only 1 g l–1 sucrose was supplied. However, the cells in this low sucrose medium in the cold were not sugar-starved, indicating that this did not explain the failure of the cells to acclimate when grown in the cold environment. Ten g l–1 sucrose supplied to cells grown in the warm (25 °C) induced acclimation to freezing and up-regulation of expression of blt4.9 and dhn1 mRNAs. Osmolality of the medium did not explain this. Thirty g l–1 sucrose induced yet higher levels of freezing tolerance and of blt4.9 and dhn1 mRNAs in cultures grown in either the cold or the warm environment. The results implicate sugars in the regulation of cold acclimation
Key words: Cold-acclimation, freezing-tolerance, gene expression, Hordeum, sugars.
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Introduction |
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How plants sense cold is unknown. A fall in temperature can reduce the fluidity of membranes, and this appears to be an effective direct sensor of cold in cyanobacteria (Nishida and Murata, 1996; Vigh et al., 1993; Suzuki et al., 2000). Another possibility is that, in light, photosynthetic cells may sense cold through an effect on photosystem II excitation pressure (Gray et al., 1997; Huner et al., 1998). Molecular studies have shown that cold acclimation in higher plants is complex involving multiple regulatory pathways (Fowler and Thomashow, 2002), furthermore that while some of the signal transduction pathways interact, others are independent (Xin and Browse, 2000). Thus more than one sensor of cold is possible.
Soluble carbohydrates have a key regulatory role in carbohydrate metabolism (Koch, 1996). They can also control events which extend well-beyond carbohydrate metabolism, such as regulating vascular differentiation (Jeffs and Northcote, 1967). Sugar-sensing and sensing of the environment appear to be part of a complex regulatory web (Gibson, 2000). In relation to cold, high sugar supply induces fructan accumulation, which is also a usual response of grasses to cold (Winter et al., 1994).
Accumulation of soluble carbohydrates is one of the best-known responses of plants to cold. It begins early during the response to cold. The soluble carbohydrate content of grasses can undergo a 10-fold increase within 8 h of transfer from a warm to a cold environment (Pollock, 1984). On the other hand, the time-scale of increase in freezing tolerance and stress-gene-expression in barley is much longer (Dunn et al., 1994; Pearce et al., 1996), indicating that changes in sugar supply may precede acclimation and cold-induced gene expression. In Arabidopsis thaliana, which acclimates much more rapidly than barley, sugar accumulation is detectable within 2 h from transfer to cold, when the increase in stress-gene expression is only just detectable, and precedes measured increase in freezing tolerance (Wanner and Juntilla, 1999).
There could be a causal connection between the accumulation of sugars and freezing-tolerance, because feeding soluble carbohydrates to plants or cultured cells induces freezing tolerance (Tumanov and Trunova, 1957; Steponkus and Lanphear, 1967; Tumanov et al., 1968; Leborgne et al., 1995; Travert et al., 1997). However, these relationships between soluble carbohydrates and freezing tolerance do not necessarily indicate that soluble carbohydrates have a regulatory role. Soluble carbohydrates could function directly to help confer freezing-tolerance through colligative and non-colligative effects (Levitt, 1980; Strauss and Hauser, 1986; Crowe et al., 1992; Travert et al., 1997). In addition, sugars may have a nutritional role during acclimation to cold (Trunova, 1982) and also during recovery from freezing-stress (Eagles et al., 1993).
Thus, in order to test for a possible regulatory role for sugars in acclimation without confounding it with these direct functional roles, it is necessary to show that, as well as affecting freezing-tolerance, sugars also modify the expression of stress-response genes, choosing genes which have no role in carbohydrate metabolism. Individual cold-response genes are not expressed in all tissues (Pearce et al., 1998). However, non-specific lipid transfer proteins (nsLTPs) are expressed in cell cultures (Meijer et al., 1993). Therefore, tests were made for the expression of blt4.9, a nsLTP gene sequence expressed in the barley crown in response to cold (White et al., 1994). A dehydrin sequence expressed in response to drought was also included, to represent genes expressed in response to other stresses (dhn1: Choi et al., 1999).
Any regulatory role sugars might have is unlikely to be exerted at the molecular level by the overall tissue concentration of sugars. When quantities of sugars are accumulated in tissues, a significant part is sequestered in cell vacuoles, and sugars in the vacuole could dominate the estimated concentration in the tissue, which consequently does not give a guide to the concentration in other subcellular compartments. Two types of sugar sensor are known which regulate carbohydrate metabolism and sugar uptake, respectively, and neither are located in the vacuole: hexokinase (Jang et al., 1997), which is located in the cytosol, and sugar sensors that are embedded in the plasma membrane (Lalonde et al., 1999).
Exposure to cold or freezing can increase apoplastic concentrations of soluble carbohydrates in cereals (Livingston and Henson, 1998). Therefore, the hypothesis that the extracellular concentration was a regulator of acclimation was tested. This may be particularly relevant to sink organs, which depend upon a supply of sugars from source organs. Sink structures essential for plant survival, such as crowns, contain a variety of young and mature organs and tissues. Therefore, in this first test of the hypothesis a barley cell culture was used as a simpler system that also provided a convenient means to supply sugars directly to the cells, replacing the culture medium daily to maintain the extracellular concentration of sugars.
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Materials and methods |
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Culture methods
Embryogenic callus cultures from immature embryos of Hordeum vulgare L. cv. Igri were established at 25 °C (in the dark) on a modified MS medium (Jahne et al., 1991) containing 30 g l–1 sucrose for stock cultures (standard medium). Liquid culture was in 100 ml of the medium in 250 ml conical flasks on a rotary shaker running at 90 rotations min–1 with a 2.5 cm horizontal throw. Treatments comprised dark environments of 25 °C or 6/2 °C (10/14 h) in test media that were the same as the standard medium except that they could contain a different sucrose concentration (BDH/Merck Analar grade). During the experiments medium was decanted from the culture vessels and replaced with fresh medium at the end of each day.
Experimental design
Both experiments were preceded by a period in which the cells were batch cultured, (therefore without daily medium replacement). The batch cultures were established by transferring 
10 g fresh weight (1 g dry weight) of cells to 100 ml of standard medium and growing the cells for 14 d without medium replacement. Typically, by the end of batch culture nutrients in the medium would be considerably depleted (Rose et al., 1972).
In experiment 1 the cells were grown in the warm (25 °C) throughout. They were transferred from batch culture, and therefore from depleted medium, to medium containing the standard medium concentrations of all nutrients except sucrose, and either high, intermediate or low concentrations of sucrose. This would determine whether sucrose or some other nutrient, without cold, could affect acclimation. The treatment media were replaced daily. The sucrose concentrations tested were: (a) 1 g l–1, 10 g l–1 or 30 g l–1 sucrose for 5 d, or (b) 2 g l–1, 10 g l–1 or 30 g l–1 sucrose, or 10 g l–1 sucrose plus 10.6 g l–1 mannitol, for 10 d.
Two factors were tested in experiment 2: the effect of (a) culture in high or low sucrose medium after transfer from a high sucrose medium, all in the warm (25 °C) (first stage), and (b) transfer from warm to cold (6/2 °C) while cultured throughout in either a high or low sucrose medium (second stage). The cells were first grown in batch culture for 14 d in the warm as in experiment 1. First stage: the cells were then transferred to fresh standard medium (therefore containing 30 g l–1 sucrose) and grown for 7 d in the warm, during which the medium was replaced daily in order to avoid substantial depletion of the sugar content of the medium. The cells were then transferred to fresh medium containing either 1 g l–1 or 30 g l–1 sucrose and culture was continued for 5 d in the warm with daily medium replacement. At this point samples were taken for analysis and the remaining cultures were used to continue the experiment. Second stage: the cells were then transferred to the cold and continued to receive daily replacement of the same medium as they had in the warm for 5 d or 10 d.
The rate of growth was measured during daily medium replacement to test if daily replacement of medium would supply sufficient sugars at low as well as high concentrations to sustain this rate. The amounts of embryogenic callus used to set up the cultures were unavoidably variable and thus the variance in estimates of growth was large. The tests indicated, at 25 °C, a mean increase (±SE) in fresh weight of 14.0±3.5% d–1 (a fresh weight doubling time of 5 d) and no significant difference between cultures supplied with 1 g 1–1, 10 g 1–1 or 30 g 1–1 sucrose. Growth in 6/2 °C was too slow to be measured accurately.
Test of freezing tolerance
Clumps of cultured cells (callus pieces) were filtered from the liquid culture media, dabbed with tissue paper to remove residual medium (centrifugation was not used for this because it distorts and compresses the cells: Reaney et al., 1996) and placed in tubes immersed in an oil bath. Ice was added to the tubes at –1.5 °C to initiate freezing. The tubes were then cooled at 6 °C h–1 to the first test temperature, held at that temperature for 45 min, and samples removed., the remaining tubes were cooled to the next test temperature and the process repeated. After thawing, the callus pieces were placed in 50 mM HEPES-NaOH, pH 7.4, containing 0.5% 2,3,5-triphenyl tetrazolium chloride (TTC) at room temperature for 1.5 h. The sample number was 15 at each freezing temperature, each sample comprising several callus pieces with a collective volume of 0.5 cm3. Viability assessment was based on the intensity of the red colour formed, on a scale of 0 (no colour; dead) to 10 (strongly and uniformly coloured; alive). The data were normally distributed. LT50 was determined by interpolation, as the temperature corresponding to 5 on the scale used. Analysis of variance was used to determine significant differences between treatments based on the TTC scores at each freezing temperature.
Extraction and analysis of soluble carbohydrates and free amino acids
Soluble carbohydrate and free amino acid extracts from cells were made using the method of Pollock and Jones (1979). Extracts or medium samples were loaded onto a Dionex PA100 guard column and separated on a PA100 column, eluting with 1 mM NaOH and the sugars were quantified using a pulsed amperimetric detector (Dionex Corp., California). Higher oligomeric and polymeric soluble carbohydrates such as fructans would also have been detected if present, though different types would not have been efficiently separated. Free amino acids were analysed, using 
-amino butyric acid as the internal standard, on a Waters Pico-Tag Amino Acid Analysis System using reverse phase HPLC (Waters Corp., Massachusetts). Medium osmolality was measured using an Osmomat 030 (Gonotec, D1000, Berlin 62, Germany).
Extraction and northern analysis of RNA
RNA was extracted from callus using a small-scale method (Wadsworth et al., 1988) using a high pH extraction buffer (Prescott and Martin, 1987). Ten µg of denatured total RNA was loaded per well and separated in a 1.2% agarose-formaldehyde gel and blotted onto Nytran-plus membrane (Schleicher and Schuell, Dassel, Germany) and fixed using an XL-1000 UV crosslinker (Spectronics Corp., New York). 32P-labelled probes were prepared by random priming. The digoxigenin-labelled EF1- sequence (blt63) riboprobe was prepared as in Pearce et al. (1998). High stringency washes (final wash 0.1x SSC and 0.1% SDS at 60 °C for 30 min) were given after hybridization with the 3'-end of blt4.9 probe (between bases 2408 and 2683 in the gDNA sequence shown by White et al., 1994) or full-length dhn1 probe. Medium stringency washes (final wash 1x SSC and 0.1% SDS at 60 °C for 30 min) were given after hybridization with the probe blt63 for elongation factor 1- (EF1-) sequences. This was used as a constitutively-expressed control sequence. Clones of a dhn1 and an EF-1 sequence (blt63) were provided by Dr TJ Close and Professor MA Hughes, respectively.
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Results |
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Freezing tolerance was strongly affected by the concentration of sucrose supplied in the medium. In the warm environment 1 g l–1 sucrose gave a low level of freezing tolerance whereas 30 g l–1 sucrose gave a high level of freezing tolerance; 2 g l–1 and 10 g l–1 sucrose gave levels of freezing tolerance significantly above the former and significantly below the latter (Table 1, experiment 1). When 1 g l–1 sucrose and 30 g l–1 sucrose were tested in cultures grown in the cold their effect was similar to in the warm (Table 1, experiment 2).
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Sucrose concentration had a much larger effect than cold on level of freezing tolerance (Table 1, experiment 2). The difference in freezing tolerance caused by using 30 g l–1 sucrose compared to 1 g l–1 sucrose was 10 °C for cells grown in the warm and 12 °C for cells grown in the cold. However, the difference in freezing tolerance between cells grown in the warm and those then transferred to the cold was only 1 °C for cells grown with 1 g l–1 and 3 °C for cells grown with 30 g l–1.
When either 25 °C or 6/2 °C was used as culture temperature, the blt4.9 and dhn1 mRNA signals were undetectable with 1 g l–1 sucrose (except for a faint trace for the dhn1 signal in the 10 d sample) and high with 30 g l–1 sucrose; the mRNA signals were intermediate with 10 g l–1 sucrose at 25 °C (Fig. 1). By contrast, the constitutively-expressed elongation factor 1 sequence gave detectable signals at all sucrose levels and both temperatures, though the levels were higher at the higher sucrose levels (Fig. 1). The increased level of expression of this sequence is consistent with the general up-regulation of metabolism during acclimation to cold (Guy, 1990). The difference between the highest and lowest EF-1 mRNA levels was much smaller than for blt4.9 and dhn1 mRNAs, indicating that expression of the two latter stress-response transcripts was differentially up-regulated compared to the representative constitutive sequence. In the barley crown, expression of blt4.9 is up-regulated by cold, but not by drought (White et al., 1994). This indicates that the up-regulation of expression of this sequence by sugars may not be due to the sugar’s osmotic effect.
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The result was the same whether cells had been transferred to these concentrations of sucrose from a nutrient-depleted medium (experiment 1) or a high sucrose medium (experiment 2) (Table 1). Experiment 2 included a period of prior culture with daily replacement of medium containing 30 g l–1. Cells that continued to be cultured in 30 g l–1 sucrose had a high level of freezing tolerance and of blt4.9 and dhn1 expression. However, cells transferred from the 30 g l–1 sucrose medium to the 1 g l–1 sucrose medium acquired a much lower level of freezing tolerance and undetectable expression of the two transcripts. Thus lowering sucrose concentration down-regulated acclimation. On the other hand, experiment 1 was initiated by taking cells at the end of 14 d batch culture without daily replacement of medium, by which time all medium constituents would have been considerably depleted (Rose et al., 1972). Transfer to high sucrose concentrations then caused a high level of freezing tolerance and blt4.9 and dhn1 transcript expression, whereas transfer to 1 g l–1 sucrose did not.
Neither the concentration of sugars supplied in the fresh medium nor the concentration remaining in the medium at the time acclimation was measured, exactly indicate what concentration controlled acclimation. This is because the concentration in the media changed during each day, between the daily replacements of medium (Fig. 2). Regulation would take time to have an effect (in whole barley plants, one day to be detectable and several days to be substantial: Pearce et al., 1996). Therefore, if this hypothesis is correct, freezing tolerance and gene expression measured at the end of 5 d or 10 d of daily medium replacement would have been a consequence of previous culture conditions, and not an instantaneous consequence of the medium composition at the time of sampling.
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By the time sampling was done, 1 d after the last replacement of medium, most of the sucrose supplied to the cultures in 1 and 10 g l–1 sucrose in the warm had been consumed. However, as rates of consumption would be slower in the cold, more remained unconsumed in the cold (Fig. 2). In the 10 g l–1 sucrose culture in the warm the concentration of remaining sugars declined to 0.2 g l–1 by the time the culture was taken for analysis (Fig. 2). However, since medium was replenished daily, the average during the previous days would have been much higher, whereas the average in the cultures supplied with 1 g l–1 would have remained low.
The composition of sugars in the medium changed during culture. The sugars that remained in most media were glucose and fructose (Fig. 2), indicating extensive hydrolysis of the sucrose supplied. The exception was the cultures in the cold supplied with 30 g l–1, in which a much lesser proportion of the sucrose was hydrolysed.
Therefore, the effect of fructose and glucose on freezing tolerance was also tested. When cells were grown at 25 °C with daily replacement of medium containing 30 g l–1 of glucose, fructose or sucrose, LT50 values were –6 °C, below –15 °C, and –14 °C, respectively. This indicates that supplying fructose in the medium was as effective as sucrose.
Cryoprotection did not explain the results. Although residual medium was removed by dabbing with tissue before the freezing test, some medium would have remained around the cells. It would be these sugars remaining at the time the cells were taken for the freezing test (and not the earlier concentrations) that would have direct cryoprotective effects. However, the concentration of sugars remaining in the medium by the time that the cells were harvested for the frost test was much lower with the 10 g l–1 sucrose treatment in the warm than with the 1 g l–1 sucrose treatment in the cold (Fig. 2). However, the former gave a much higher level of freezing tolerance than the latter (Table 1). This indicates that direct cryoprotection could not explain freezing tolerance in the 10 g l–1 sucrose treatment. Similarly, the differences in gene expression could not be explained by direct cryoprotection.
It was necessary to test whether the results could be explained by an osmotic effect of treatments. The 10 g l–1 sucrose medium was supplemented with 10.6 g l–1 mannitol to give the same osmolarity due to the sucrose plus mannitol in the medium as when 30 g l–1 sucrose was used. Unexpectedly, this reduced freezing tolerance (Table 1). The tetrazolium staining of the sucrose plus mannitol-grown cells used as controls in the frost-test was normal, indicating mannitol treatment was not lethal. A possible explanation for the results is that mannitol, or mannose formed from it, which can down-regulate expression of leaf genes (Jang and Sheen, 1994), had an adverse regulatory effect on carbohydrate metabolism or, more directly, on factors affecting acclimation. If sucrose or fructose have a regulatory role in acclimation, then competitive inhibition of their effect by polyols is not impossible. In other experiments sorbitol and mannitol supplied to A. thaliana over days had adverse effects, indicating that polyols were possibly not suitable for testing osmotic effects on cold-acclimation during prolonged culture (data not shown).
Therefore, direct measurements of the osmolality of the culture media were made, thus avoiding reliance on polyols as the means to test if sugar regulation of acclimation was osmotic. The results indicated that acclimation was not explained by the osmotic potential of the media. The freezing tolerance and levels of blt4.9 and dhn1 transcripts were much higher in cells cultured in 10 g l–1 sucrose in the warm than in cells cultured in 1 g l–1 sucrose in either the warm or the cold (Table 1; Fig. 1). Osmolality of fresh medium, before use in culture, differed by 30% between the 1 g l–1 (0.10±0.0 Osm kg–1) and 10 g l–1 sucrose-media (0.13±0.0 Osm kg–1). After culture in the warm environment, however, the difference in osmolality between the 1 g l–1 and 10 g l–1 sucrose-media had disappeared, whereas the osmolality of the 1 g l–1 sucrose medium in the cold was 33% higher than that of the 10 g l–1 sucrose medium (Table 2). In both comparisons, particularly the latter, the average difference in osmolality during culture would have been small.
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It was necessary to test if the failure of the cells grown in 1 g l–1 sucrose to acclimate in response to cold was due to sugar starvation. When the cultures were supplied with 1 g l–1 sucrose in the cold, sugars (predominantly glucose and fructose) were still present in the medium that remained after the period of culture (Fig. 2). Thus sugars were not exhausted from the low sucrose medium in the cold.
The cells contained glucose, fructose and sucrose (Fig. 3). However, higher molecular weight soluble carbohydrates such as fructans were not present. When supplied with 1 g l–1 sucrose in the cold, the cells accumulated higher concentrations of total sugars than were present in the medium, and total sugar concentra tion in the cells approximately doubled between the fifth day and the tenth day of this treatment (Fig. 3). This evidence of accumulation in cells indicates that, in this treatment (1 g l–1 sucrose in the cold), there was an excess of sugars over the amount that could be metabolized by the cells.
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If sugar starvation were occurring in the low-sugar cultures then cellular energy levels would be at risk. Typically, sugar-starved cells would hydrolyse proteins to release free amino acids and these could then provide organic acids to the respiratory pathways. As a consequence, during sugar-starvation the level and composition of the free amino acid pool would change. In the early stages of the consumption of proteins in this way, concentrations of certain free amino acids would rise, particularly asparagine but also serine, aspartic acid and glycine. Later, as the available proteins became consumed, the concentrations of all free amino acids would fall to very low values (Brouquisse et al., 1992).
No cultures showed the latter symptom (Table 2), thus sugar starvation was never advanced. In most cultures, alanine and glutamic acid were the most abundant free amino acids (mean mol% of total amino acids in cells from the different treatments, ±standard deviation: 29±11 and 17±9, respectively), followed by proline (Fig. 4). The cells grown in 1 g l–1 sucrose in the warm contained a low concentration of sugars (Fig. 3) and asparagine was abundant, indicating sugar-starvation symptoms could be revealed in these experiments by measurements of free amino acids. Interestingly, cells grown in 10 g l–1 sucrose in the warm environment also contained a high concentration of asparagine, yet were able to acclimate to cold, possibly indicating that the inception of starvation may not totally prevent acclimation. For comparison, mineral starvation in barley did reduce acclimation somewhat, but, again, significant acclimation still occurred (Pearce et al., 1996).
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The effect of 1 g l–1 sucrose in the cold was critical, since cells in this treatment did not acclimate despite being transferred from the warm to the cold environment. Asparagine, serine, aspartic acid, and glycine were present in low concentrations, similar to those in the cells cultured with high sugar supply (Fig. 4; and concentrations of other free amino acids were also similar—not shown). This, together with the evidence of reserves of sugars in the medium and cells (Figs 2, 3), indicates that the cells grown in 1 g l–1 sucrose in the cold were not suffering sugar starvation.
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Discussion |
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When the cells were grown in the cold they required high extracellular concentrations of sugars in order to express high freezing tolerance and high stress gene expression. Sugar starvation did not occur at low sugar concentrations in the cold, thus this could not explain the requirement for a high concentration of sugars. The supply of high sugar concentrations caused acclimation in the warm environment. Thus cold was not required for acclimation by these cultures. The simplest explanation was that the sugars supplied in the medium regulated acclimation, but this did not seem to be due to an osmotic signal, and therefore indicated a more direct regulatory role for sugars.
It may be argued that, since the cells grown in the warm in a normal plant cell culture medium have high freezing tolerance, that must be the natural or ‘base’ level of freezing tolerance for those cultured cells. However, a ‘base’ level that is specific to one set of culture conditions is not a true base level. In the present experiments, the cells grown in the cold in low sucrose were not sugar-starved and yet had a low freezing tolerance. Hence high freezing tolerance was not a base level for these cultured cells.
Plant cell culture media typically contain high nutrient concentrations and during batch culture and transfer to fresh medium the cells are exposed to repeated cycles of decline in nutrient concentrations followed by transfer to high concentrations again (Street, 1973; Rose et al., 1972). However, the effect of sucrose appeared to be independent of whether the treatment given created an up-regulation or a down-regulation of acclimation. In the field, plants are exposed to cold that varies in intensity with time, and their level of acclimation tracks this (Sakai and Larcher, 1987). Speculatively, the regulatory role of sugars in acclimation could be in relation to this long-term modulation of level of acclimation.
The concentrations that caused high levels of acclimation corresponded to concentrations normally provided in freshly prepared plant cell culture media. Thus these experiments could help to explain how cell cultures acclimate to cold. However, the concentrations used here also corresponded to concentrations found in the apoplast of oats, before and during cold acclimation (Livingston and Henson, 1998). Thus the experiments are also relevant to the acclimation of tissues in the whole plant.
Apoplast concentrations of sugars can be high during acclimation. Cereals acclimate in two stages: cold (low positive temperatures) induces a certain amount of freezing tolerance, but subsequent exposure to freezing increases tolerance yet further (Trunova, 1965), including increasing the level of expression of cold-induced genes (Pearce et al., 1996). Livingston and Henson (1998) detected glucose, fructose, sucrose, and fructans in the apoplast of crowns of control oat plants and in oat plants exposed to cold alone or to cold followed by freezing (–3 °C). They ascribed much of the soluble carbohydrates in the apoplast of the crown of control and cold-grown plants to leakage from cells during the extraction of apoplastic fluid. However, the fluid from the plants exposed to –3 °C had a much higher concentration, of 30 g l–1 for sucrose plus fructose, and was not contaminated. We found that 30 g l–1 sucrose or fructose gave high levels of acclimation. Livingston and Henson (1998) used guttate to give an uncontaminated indication of sugars in the apoplast of leaves from cold-acclimated plants. This contained 3 g l–1 of sucrose plus fructose. We found that 2 g l–1 could induce partial acclimation to freezing. Furthermore, in both weight and molar terms, the quantity of all carbohydrates in the guttate exceeded the quantity provided by the 10 g l–1 sucrose treatment used here, which gave a similar level of acclimation to that found in whole barley plants exposed to cold. Thus it is reasonable to suggest that extracellular concentrations of sugars are a factor in cold acclimation of tissues in the whole plant.
Whereas the concentration of sugars supplied in the medium was related to the level of frost-tolerance and of stress-gene expression, the level of sugars within the cells was not. This is not counter to the hypothesis proposed, which addresses the extracellular concentration and the supply of sugars to the cells, not the concentration within them. Nor is it counter to the widely-accepted idea (see earlier references) that sugar accumulation has a non-regulatory role in acclimation to cold, which would function alongside any regulatory role. We assume that any sugar-sensing would either occur during initial metabolism in the cytosol (Jang et al., 1997), or possibly at the plasma membrane (Lalonde et al., 1999), and that sugars accumulated in the vacuole may not be involved.
Expression of sucrose phosphate synthase (SPS) is up-regulated by cold (Guy et al., 1992; Reimholz et al., 1997), and cold activates SPS (Hill et al., 1996). It might be thought that a cold signal must therefore precede any sugar signal. This could be wrong. Cold-acclimation requires the allocation of carbohydrates between constitutive processes and processes that support the acquisition of winter-stress-tolerance. Cold up-regulates expression of sucrose synthase (SS) as well (Déjardin et al., 1999). SS can help channel carbohydrate into primary metabolism, whereas SPS can help shift the flux of carbohydrates towards the sequestration of soluble sugars. The control of allocation between these alternative uses must be important in cold acclimation, and it would not be surprising if sugar supply helped exert it.
The levels of freezing-tolerance achieved in the cultures when grown with 10 g l–1 or 30 g l–1 sucrose in the warm or cold (LT50 values from –10.2 °C to –17.5 °C: Table 1) were in the same range as those found within the crown of the whole plant when acclimated to controlled environments of 6/2 °C or +4/–4 °C or to a natural frosty winter period: LT50 values of –10.5 °C, –17.8 °C and –14.5 °C, respectively (Pearce et al., 1996). RNA extracted from crowns of whole plants grown in 6/2 °C gave similar blt4.9 and dhn1 mRNA signals to those obtained with 10 g l–1 sucrose in liquid cultures grown at 25 °C (Fig. 1). Thus, differences in sugar supply over the range tested here could explain the control of cold-acclimation in whole barley plants. However, there were differences in the details of cold-acclimation between the cell cultures and whole plants. Thus fructans, which accumulate in Poaceae during cold-acclimation, were not present. On the other hand, proline, which commonly accumulates during cold-acclimation, was an abundant free amino acid in most treatments including in cells in the 1 g l–1 treatment in the cold, which did not acclimate.
Plants of A. thaliana transferred to cold and dark, and not accumulating sugars, did not acquire freezing-tolerance (Wanner and Juntilla, 1999), just as the cell cultures in the cold with a low sugar supply did not in this work. However, whereas this was found to be associated with a lack of cold-induced expression of the test genes used here, those authors found that cold-inducible genes were expressed (Wanner and Juntilla, 1999). The explanation for this difference in finding is unclear, but presumably it could be an effect of differences in physiology between leaf cells, a source organ, and the cultured cells, acting as a sink.
Most previous studies that have indicated a role for sugars in acclimation to cold, found that cold was also necessary (Tumanov and Trunova, 1957; Steponkus and Lanphear, 1967; Wanner and Juntilla, 1999). However, the experiments were with whole plants or plant parts. On the other hand others have found, as we did, that in cell cultures sugars without cold could induce freezing tolerance (Leborgne et al., 1995; Travert et al., 1997).
A striking and informative example is from Chen and Gusta (1982). The control (warm-grown) brome grass cell cultures had an LT50 of –13 °C. During subsequent growth in the cold, freezing-tolerance increased to about –18 °C and then fell to –15 °C. Thus the effect of cold was small compared to the control level of freezing tolerance. Furthermore, the increase in the level of acclimation induced was unstable. Later experiments from the same laboratory gave control freezing tolerance for the same cultures as usually near –8 °C (Robertson et al., 1987), though a value of –11 °C was recently reported (Robertson et al, 1994). Robertson et al. (1994) also reported that the freezing tolerance of non-acclimated young brome grass plants was –3 °C, and that after acclimation to cold for 14 d their freezing tolerance was –8 °C. Thus, in the absence of cold, the cultured brome grass cells already had a freezing tolerance comparable to or higher than in the acclimated young plants. Clearly, consistent with our findings, this is an indication that cold is not essential for a significant level of acclimation in cell cultures.
The reason for the difference in behaviour between whole plants and organs, which require cold as well as sugars, and cell cultures, which require only sugars, is not clear. The possibility has not been discounted that some other medium component, or the constitutively present intracellular proline (whose concentrations are similar to those found in freezing-tolerant A. thaliana: Xin and Browse, 2000), ‘primes’ the cells to acclimate in response to sugar supply.
However, there are other possibilities. The difference in size, organization, and tissue composition between the whole plant and cell culture could be important. The whole plant can behave in a complex way, for example, development of leaves in the cold changes regulation, since it relieves the initially suppressive regulatory effect of sugars on photosynthesis (Strand et al., 1997). It is also clear that different tissues within an organ have a different immediate response to cold, since they express different gene sequences (Pearce et al., 1998). If the sensing of stress or the operation of signal transduction pathways differed in different tissues and organs, the whole plant could display a combination of requirements for acclimation which no single tissue or a cell culture might display. The experiments here do not show how a plant would respond as a whole to cold, which is too complex for a cell culture to mimic, but they do implicate sugars in a regulatory role in acclimation.
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SRTA gratefully acknowledges the financial support of the Islamic Republic of Iran. The support of the Natural Environment Research Council and of the Biotechnological and Biological Sciences Research Council and the technical assistance of Sue Patterson, Bob Nicholson and Hilary Mason are also gratefully acknowledged.
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