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The Plant Journal (2000) 23(6), 785±794 Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane ¯uidity BjoÈrn LaÂrus OÈrvar*,², Veena Sangwan, Franz Omann and Rajinder S. Dhindsa Department of Biology, McGill University, 1205 Avenue Docteur Pen®eld, MontreÂal, QueÂbec H3A 1B1, Canada Received 5 May 2000; revised 23 June 2000; accepted 30 June 2000. *For correspondence (fax +35 457 07111; e-mail [email protected]). ² Present address: Technological Institute of Iceland, Keldnaholt, IS-112 Reykjavik, Iceland. Summary Many plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non-freezing temperatures at the onset of winter. CA is associated with gene expression that requires transient calcium in¯ux into the cytosol. Alfalfa (Medicago sativa) cells treated with agents blocking this in¯ux are unable to cold-acclimatize. Conversely, chemical agents causing increased calcium in¯ux induce cold acclimatization-speci®c (cas) gene expression in alfalfa at 25LC. How low temperature triggers calcium in¯ux is, however, unknown. We report here that induction of a CA-speci®c gene (cas30), calcium in¯ux and freezing tolerance at 4LC are all prevented by cell membrane ¯uidization, but, conversely, are induced at 25LC by membrane rigidi®cation. cas30 expression and calcium in¯ux at 4LC are also prevented by jasplakinolide (JK), an actin micro®lament stabilizer, but induced at 25LC by the actin micro®lament destabilizer cytochalasin D (CD). JK blocked the membrane rigidi®er-induced, but not the calcium channel agonist-induced cas30 expression at 25LC. These ®ndings indicate that cytoskeleton re-organization is an integral component in low-temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidi®cation and calcium in¯ux in CA. Keywords: cold acclimatization, signalling, actin, membrane ¯uidity. Introduction Cold acclimatization (CA) in plants, resulting in acquisition of increased freezing tolerance, requires exposure to low but non-freezing temperatures over a periods of days or weeks (Levitt, 1980). Transient in¯ux of Ca2+ into the cytosol is an early event during CA (Knight et al., 1991; Monroy and Dhindsa, 1995). Blocking this Ca2+ in¯ux prevents cold-induced gene expression and development of freezing tolerance in alfalfa (Medicago sativa) cells (Monroy et al., 1993) and in Arabidopsis (Tahtiharju et al., 1997). Conversely, chemical agents that increase Ca2+ in¯ux, such as the Ca2+ channel agonist Bay K 8644 or the Ca2+ ionophore A23187, induce CA-speci®c (cas) gene expression in alfalfa cells at 25°C (Monroy and Dhindsa, 1995). These results demonstrate that cold-induced in¯ux of Ca2+ is an integral component of the low-temperature signalling cascade during CA. However, the nature and temporal sequence of events leading to this Ca2+ in¯ux are largely unknown. A direct and perhaps the earliest effect of an alteration of temperature on cells is the change in ¯uidity of their ã 2000 Blackwell Science Ltd membranes (Levitt, 1980). A decrease in temperature lowers membrane ¯uidity, whereas its increase enhances membrane ¯uidity (Alonso et al., 1997; Mejia et al., 1995). Previous studies (Murata and Los, 1997) have suggested that the plasma membrane acts as the primary sensor of temperature change through dynamic changes in its physical state. Rigidi®cation of plasma membranes by Pd-catalysed hydrogenation leads to the induction of the cold-inducible gene, desA, encoding a fatty acid desaturase in the cyanobacterium, Synecocystis (Vigh et al., 1993). It is not known if, and how, membrane ¯uidity modulates ion channel activity. Mazars et al. (1997) have shown that cold-induced Ca2+ in¯ux in tobacco protoplasts was stimulated by disruption of microtubules and actin micro®laments. Recent studies have also demonstrated roles for actin micro®laments in K+ channel activity in stomatal opening (Hwang et al., 1997), and in mechanical stress signalling in plants (Morelli et al., 1998). Although Ca2+ channel proteins have yet to be characterized in plants, a plant gene (AKT1) encoding a putative K+ channel has 785 786 BjoÈrn LaÂrus OÈrvar et al. been cloned (Sentenac et al., 1992). The deduced protein contains ankyrin-like repeats suggesting that ion channel proteins in plants may interact with the cytoskeleton (Sentenac et al., 1992). The above observations led us to explore whether changes in membrane ¯uidity and re-organization of the actin cytoskeleton have a role in the early stages of lowtemperature signalling in plant cells. We also investigated whether these events are linked to Ca2+ in¯ux and CA. We have used the transcript level of a CA-speci®c gene (cas30) and the development of freezing tolerance in alfalfa cell suspension cultures as end-point markers in low-temperature signalling. Our results suggest that cold signalling in plants may proceed from membrane rigidi®cation through actin micro®lament re-organization to Ca2+ in¯ux and CAspeci®c gene expression, eventually leading to CA and increased frost tolerance. Results Chemical modulation of membrane ¯uidity in alfalfa protoplasts Both DMSO and benzyl alcohol (BA) have been routinely used as a membrane rigidi®er and ¯uidizer, respectively. Fluorescence polarization of 1,6-diphenyl1,3,5-hexatriene (DPH) was used to con®rm the effects of these agents on alfalfa protoplasts, but the polarization index p is inversely related to membrane ¯uidity (Choi and Yu, 1995). The data presented in Table 1 show that DMSO treatment reduces membrane ¯uidity at 25°C as demonstrated by an increase in the polarization index p. Conversely, pre-treatment of protoplasts with BA inhibits the cold-induced increase in p, or a decrease in membrane ¯uidity. Membrane ¯uidization inhibits cold signalling in alfalfa cells To test the hypothesis that membrane ¯uidity plays a fundamental role in low-temperature signal transduction in alfalfa, transcript accumulation of a CA-speci®c gene, cas30, and the development of freezing tolerance in alfalfa cell suspension cultures were used as end-point markers. The expression of cas30 is speci®cally induced by cold treatment and is not responsive to other stresses investigated (Mohapatra et al., 1989), and therefore serves as a reliable end-point marker for CA. Cold treatment causes a rapid and reversible accumulation of cas30 transcripts (Figure 1; data not shown). BA, a well-established membrane ¯uidizer (Carratu et al., 1996; Kitagawa and Hirata, 1992), was used to counter cold-induced membrane rigidi®cation. When cells were pre-treated with BA for 1 h at 25°C before being incubated at 4°C for 6 days in the presence of the alcohol, the cold-induced cas30 expression was inhibited at all concentrations tested (Figure 2a). BA treatment also strongly inhibited the development of freezing tolerance (Figure 2b) without affecting cell viability (data not shown). Similar results were obtained by treating cells with ethanol, another membrane ¯uidizer (Mrak, 1992) (data not shown). To investigate whether lowering of the cas30 transcript levels by BA treatment is due to a decrease of transcript levels in general, transcript levels for the replacement histone H3.2 protein and for the putative L5 ribosomal protein from alfalfa were determined. The H3.2 (Robertson et al., 1996) and L5 (Asemota et al., 1994) transcripts are constitutively expressed in alfalfa. Both the L5 and H3.2 transcript levels, which are not detectably affected after 48 h CA or during deacclimatization (Figure 1), are also not affected by BA treatment (Figure 2a). Thus the effects of BA on cas30 transcript levels do not re¯ect a general down-regulation of transcription. CA in alfalfa suspension cells involves a rapid in¯ux of Ca2+ into the cytosol as determined by 45Ca2+ cation exchange chromatography as previously described (Monroy and Dhindsa, 1995). Isolated protoplasts were used to measure Ca2+ in¯ux in order to avoid complications due to Ca2+-rich cell walls, but cold-induced accumulation of cas30 transcripts also occurs in isolated protoplasts (data not shown). The source for the coldinduced Ca2+ in¯ux has been shown to be primarily apoplastic in alfalfa cells and is strongly inhibited by Ca2+ chelators and Ca2+ channel blockers (Monroy and Dhindsa, 1995). Using 45Ca2+ cation exchange chromatography (Monroy and Dhindsa, 1995), we examined the effects of 5 mM BA treatment on cold-induced Ca2+ in¯ux. This concentration of BA was used since it had no effect on protoplast viability, as determined by Evan's blue staining (data not shown), and it inhibits cold-induced membrane rigidi®cation (Table 1). Treatment of protoplasts with 5 mM BA inhibited Ca2+ in¯ux (Figure 2c). Thus, at 4°C, Ca2+ in¯ux, cas30 gene expression and freezing tolerance are all prevented by BA-induced cell membrane ¯uidization. Membrane rigidi®cation activates cold signalling in alfalfa cells at 25°C We reasoned that if reduced membrane ¯uidity mediates the cold-induced cas30 transcript accumulation, Ca2+ in¯ux and increased freezing tolerance, then reduction of membrane ¯uidity at 25°C by chemical agents should mimic these responses. Treatment of cells at 25°C with DMSO, a well-documented membrane rigidi®er (Lyman and Preisler, 1976), at concentrations as low as 1%, induced cas30 transcript accumulation (Figure 3a), but did not affect the H3.2 and L5 mRNA levels (Figure 3a). DMSO treatment at 25°C increased membrane rigidi®caã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 Early steps in cold sensing by plant cells 787 Table 1. Polarization index (p) of isolated protoplasts treated with cold, BA, DMSO, JK or CD Treatment Temperature (°C) Polarization index (p) Experiment 1 Control Control 25 4 0.0954 6 0.0073a 0.1315 6 0.0138b Experiment 2 Control Control BA (5 mM) 25 4 4 0.0825 6 0.0043a 0.1173 6 0.0122b 0.0853 6 0.0064a Experiment 3 Control Control DMSO (3%) 25 4 25 0.0870 6 0.0015a 0.1322 6 0.0048b 0.1003 6 0.0029c Experiment 4 Control Control JK (2 mM) 25 4 4 0.0921 6 0.0109a 0.1555 6 0.0136b 0.1525 6 0.0085b Experiment 5 Control CD (50 mM) 25 25 0.1084 6 0.0115a 0.1053 6 0.0102a Data are expressed as the mean 6 SD of three replicates. Values with different letters are signi®cantly different (P < 0.05) in each experiment as determined by Student's t-test. tion in isolated protoplasts (Table 1), enhanced the freezing tolerance of cells (Figure 3b) and promoted Ca2+ in¯ux (Figure 3c). Cell and protoplast viability was not affected by this chemical (data not shown). Therefore, the examined events characteristic of CA, namely cold-induced cas30 transcript accumulation, Ca2+ in¯ux and increased freezing tolerance, can be induced by membrane rigidi®cation at 25°C, or prevented at 4°C by membrane ¯uidization. Figure 1. Cas30 transcript accumulation during 48 h cold acclimatization (CA), and subsequent disappearance following de-acclimatization (DA) at 25°C. Total RNA (10 mg) was hybridized to labelled cas30 cDNA. Filters were stripped and re-probed with labelled L5 and H3.2 cDNAs. Equal loading of the gel was con®rmed by negative imaging of the RNA blot. plant actin micro®laments. Treatment with JK inhibits cold-induced cas30 transcript accumulation (Figure 4a) and Ca2+ in¯ux (Figure 4b), and lowers freezing tolerance (Figure 4c). Conversely, treatment of cells with CD caused both cas30 transcript accumulation (Figure 5a) and increased Ca2+ in¯ux (Figure 5b) at 25°C. However, CD treatment at 25°C reduced the constitutive level of freezing tolerance (Figure 5c). Cell or protoplast viability was unaffected by treatment with JK or CD (data not shown). Furthermore, membrane ¯uidity (Table 1) and transcript levels of H3.2 and L5 were apparently unaffected by either JK or CD treatment (Figure 4a and Figure 5a). Thus, Ca2+ in¯ux and cas30 expression are induced by an actin micro®lament destabilizing agent, and, conversely, inhibited by an actin micro®lament stabilizing agent. Disruption of actin micro®laments is required for cold signalling in alfalfa cells Temporal order of membrane rigidi®cation and actin micro®lament re-arrangement in cold signalling in alfalfa cells Yamamoto (1989) demonstrated that membrane rigidi®erinduced Ca2+ in¯ux in hepatocytes is accompanied by actin micro®lament re-organization. It has also been shown that cold-induced Ca2+ in¯ux in tobacco protoplasts can be enhanced (almost doubled) by chemical such as cytochalasin D (CD) that modulate actin micro®lament organization (Mazars et al., 1997). We therefore examined whether modulators of actin micro®lament re-organization mimic the effect of cold-induced rigidi®cation of the membrane on Ca2+ channels and cas30 expression. To modulate actin micro®lament organization we used CD (Cooper, 1987) and jasplakinolide (JK)(Bubb et al., 1994), both membrane-permeating chemicals that have been successfully used as a destabilizer (Hwang et al., 1997; Mazars et al., 1997; Morelli et al., 1998) and stabilizer (Mathur et al., 1999; Sawitzky et al., 1999), respectively, of The results presented above suggest that cold-induced rigidi®cation of membranes and actin micro®lament reorganization are required for Ca2+ in¯ux and cas30 transcript accumulation. However, the temporal order of these events remained to be determined. By using a combination of chemical treatments, we presumed that promoting an earlier event while blocking a subsequent one should prevent cas30 transcript accumulation. Conversely, blocking an earlier event but promoting a downstream one should result in cas30 transcript accumulation. It has been previously demonstrated that the cold induction of cas30 requires Ca2+ in¯ux (Monroy et al., 1993). The data in Figure 6 show that cas30 expression induced at 25°C by either DMSO treatment (membrane rigidi®er) or by CD treatment (micro®lament destabilizer) is inhibited by the Ca2+ chelator 1,2-bis(2-aminophenoxy)- ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 788 BjoÈrn LaÂrus OÈrvar et al. Figure 2. Membrane ¯uidization inhibits cold-triggered cas30 transcript accumulation, development of freezing tolerance and Ca2+ in¯ux in alfalfa cells. (a) Inhibition of cold-induced cas30 transcript accumulation by the membrane ¯uidizer BA. Cells were treated for 1 h at 25°C with the indicated concentrations of BA and subsequently placed at 4°C for 24 h before RNA extraction. Filters were stripped and re-probed with labelled L5 and H3.2 cDNAs. (b) Inhibition of cold-induced development of freezing tolerance by BA. Cells were treated for 1 h at 25°C with BA, then incubated for 6 days at 4°C. Freezing tolerance is expressed as the ratio (%) of BA-treated versus untreated cells. (c) BA inhibition of coldtriggered Ca2+ in¯ux. Protoplasts were treated with 5 mM BA for 30 min, followed by a 20 min incubation with 45Ca2+ at 4°C. 45Ca2+ in¯ux is expressed as the ratio (%) of BA-treated versus untreated (C) protoplasts, both at 4°C. Although 20 mM BA treatment did not affect the viability of intact cells, it reduced the viability of isolated protoplasts by 50%, whereas 5 mM had no effects (data not shown). Error bars in (b) and (c) represent the standard deviation of the mean of three and four replicates, respectively. ethane N,N,N¢,N¢-tetraacetic acid (BAPTA, Figure 6a,b). These results indicate that Ca2+ in¯ux is downstream of both membrane rigidi®cation and actin micro®lament reorganization, and further extend the results presented in Figure 3. Membrane rigidi®cation induces cas30 transcript accumulation, development of freezing tolerance and Ca2+ in¯ux in alfalfa cells at 25°C. (a) Induction of cas30 transcript accumulation by the membrane rigidi®er DMSO at 25°C. Cells were incubated for 3 h with DMSO before RNA extraction. Filters were stripped and re-probed with labelled L5 and H3.2 cDNAs. (b) Induction of freezing tolerance by DMSO. Cells were treated for 3 days with DMSO at 25°C. Freezing tolerance is expressed as the ratio (%) of DMSO-treated versus untreated cells. (c) Induction of Ca2+ in¯ux by DMSO at 25°C, compared to cold-induced Ca2+ in¯ux. Protoplasts were treated for 30 min with 3% DMSO before incubation with 45Ca2+ at 25°C. Error bars in (b) and (c) represent the standard deviation of the mean of three and four replicates, respectively. Figures 3(c) and 5(b). The DMSO-induced cas30 expression at 25°C is also inhibited by the actin micro®lament stabilizer, JK (Figure 6c). However, the induction of cas30 at 25°C by the Ca2+ channel agonist Bay K 8644, although sensitive to BAPTA, is not inhibited by JK (Figure 6d). These data demonstrate that cold-induced re-organization of actin micro®laments is located downstream of membrane rigidi®cation but upstream of Ca2+ in¯ux. ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 Early steps in cold sensing by plant cells 789 2+ Figure 4. Cold-induction of cas30 transcript accumulation, Ca in¯ux and freezing tolerance are inhibited by the actin micro®lament stabilizer jasplakinolide (JK). (a) Inhibition of the cold-induced cas30 transcript accumulation by JK. RNA was isolated from cells treated for 1 h at 25°C with JK (2 mM) or solvent vehicle alone (C; 0.15% DMSO), followed by 24 h incubation at 4°C. Filters were stripped and re-probed with labelled L5 and H3.2 cDNAs. (b) Inhibition of cold-induced Ca2+ in¯ux by JK. Protoplasts were treated for 30 min with JK (2 mM) or solvent vehicle alone (C) at 25°C before incubation with 45Ca2+ at 4°C for 20 min. (c) Inhibition of coldinduced development of freezing tolerance by JK. Cells were treated for 1 h at 25°C with JK (2 mM) or solvent vehicle alone and then incubated for 6 days at 4°C. Freezing tolerance of JK-treated cells is expressed as the ratio (%) of that of cells treated with solvent vehicle alone. Error bars in (b) and (c) represent the standard deviation of the mean of four replicates. Discussion The underlying mechanisms of low temperature sensing and signal transduction in plants are poorly understood. Here we demonstrate that, in alfalfa cells, (a) cold-induced Ca2+ in¯ux, cas30 transcript accumulation and development of freezing tolerance are inhibited by chemicals that either ¯uidize membranes or stabilize actin micro®laments; (b) chemicals that either rigidify membranes or destabilize actin micro®laments cause Ca2+ in¯ux and cas30 expression at 25°C; and (c) the role of actin cytoskeleton in low-temperature signal transduction is located downstream of membrane rigidi®cation but upstream of Ca2+ in¯ux. The importance of membrane ¯uidity in low-temperature perception has long been recognized (Levitt, 1980; ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 Figure 5. cas30 transcripts accumulation and Ca2+ in¯ux are induced by the actin micro®lament destabilizer cytochalasin D (CD). (a) CD-induced cas30 mRNA accumulation at 25°C. RNA was isolated from cells treated for 3 h at 25°C with CD (50 mM) or solvent vehicle alone (C; 0.64% DMSO). (b) Induction of Ca2+ in¯ux by CD. Protoplasts were treated for 30 min with CD (50 mM) or solvent vehicle alone (C) at 25°C before incubation with 45Ca2+ for 20 min. (c) Inhibition of cold-induced development of freezing tolerance by CD. Cells were treated for 3 days with CD (50 mM) or solvent vehicle alone at 25°C. Freezing tolerance was calculated as the percentage of that of cells subjected to solvent vehicle treatment alone. Error bars in (b) and (c) represent the standard deviation of the mean of four replicates. Murata and Los, 1997). However, its relationship to subsequent responses to cold has been unclear. Our results show that DMSO treatment induces membrane rigidi®cation and mimics the effects of cold on gene expression, Ca2+ in¯ux and cold acclimatization. Furthermore, counteracting the cold-induced membrane rigidi®cation with chemical ¯uidizers halts all examined responses to cold, suggesting that membrane rigidi®cation is not only a suf®cient but a necessary condition for cold signalling in plant cells. It should be noted, however, that although 3% DMSO has almost saturating effects on freezing tolerance, its effect on cas30 expression is not saturated. While the reasons for this are unclear, it is possible that DMSO induces a battery of cas genes, besides cas30, leading to enhanced freezing tolerance. Alternatively, DMSO may also be exerting its well-known cryoprotective effect which may or may not be related to its effect on membrane rigidi®cation. It is widely accepted that Ca2+ in¯ux into the cytosol is triggered by cold (Knight et al., 1991) and is required for 790 BjoÈrn LaÂrus OÈrvar et al. Figure 6. Cold-induced re-organization of actin micro®laments provides a link between membrane rigidi®cation and Ca2+ in¯ux. (a) Induction of cas30 transcript accumulation by DMSO at 25°C is inhibited by the Ca2+ chelator BAPTA. Cells were treated for 1 h with BAPTA (BP; 2 mM) and then for 3 h with DMSO (DM; 3%). Control cells were treated with DMSO (3%) or culture medium alone (C) for 3 h. (b) Induction of cas30 transcript accumulation by CD is inhibited by BAPTA. Cells were treated for 1 h with BAPTA (2 mM) and then for 3 h with CD (50 mM). Control cells were treated with CD (50 mM) or solvent vehicle alone (C; 0.64% DMSO) for 3 h. (c) Induction of cas30 transcript accumulation by DMSO at 25°C is inhibited by JK. Cells were treated for 1 h with JK (1 mM) and then for 3 h with DMSO (3%) at 25°C. Control cells were treated as described in (a). (d) Induction of cas30 transcript accumulation at 25°C by the Ca2+ channel agonist Bay K 8644 is inhibited by BAPTA but not by JK. Cells were treated for 1 h with either JK (1 mM) or BAPTA (2 mM) and then for 3 h with Bay K 8644 (BK; 100 mM). Control cells were treated with Bay K 8644 (100 mM) or solvent vehicle alone (C) alone for 3 h. cold-induced gene expression and development of freezing tolerance (Knight et al., 1996; Monroy and Dhindsa, 1995; Monroy et al., 1993; Tahtiharju et al., 1997). The data presented here indicate that cold-triggered Ca2+ in¯ux is downstream of membrane rigidi®cation, because the in¯ux is inhibited by membrane ¯uidization (Figure 2c) but triggered at 25°C by membrane rigidi®cation (Figure 3c). Although membrane rigidi®cation-induced Ca2+ in¯ux has not been reported in plants, DMSO-induced membrane rigidi®cation induces Ca2+ in¯ux in hepatocytes (Yamamoto, 1989) and parathyroid cells (Nygren et al., 1987). Cholesterol, another frequently used modulator of membrane ¯uidity, increases membrane rigidity and causes Ca2+ in¯ux in rabbit smooth muscle cells (Gleason et al., 1991). Thus, membrane rigidi®cation and Ca2+ in¯ux appear to be coupled. It should be noted that our data indicate little about the changes in actual concentration of Ca2+ in the cytosol and refer strictly to in¯ux of extracellular Ca2+ into the cell. The transient rise in cytosolic Ca2+ concentration due to cold shock is well established through extensive research by many researchers, notably by Trewavas and colleagues (Knight et al., 1991). In our study, treatment with CD causes both Ca2+ in¯ux (Figure 5b) and cas30 expression at 25°C (Figure 5a). Conversely, treatment with JK inhibits both these events at 4°C (Figure 3a,b). JK can simultaneously stabilize one population of actin micro®laments and destabilize another, or rapidly induce nucleation of actin polymerization, depending on the cellular domain (Spector et al., 1999). Immunochemical estimation of F-actin levels has shown that JK pre-treatment causes a dramatic increase in F-actin levels in cold-shocked cells (OÈrvar et al., unpublished data), indicating that JK stabilizes the micro®laments. However, the mechanism of CD action is quite complex. CD can cap the barbed end of actin micro®laments, severe the micro®laments and sequester actin monomers, and thus affect the abundance and organization of micro®laments (Cooper, 1987; Goddette and Frieden, 1986; Sampath and Pollard, 1991). Although latrunculin B, a powerful actin micro®lament-disrupting agent (Spector et al., 1983), has similar effects on cas30 expression as does CD (data not shown), it is also plausible that the cold-induced Ca2+ entry mimicked by CD treatment (Figure 5b), does not require depolymerizaton of micro®laments, but instead remodelling of the pre-existing network. To date, although lowtemperature disruption of the actin cytoskeleton has been demonstrated in animal cells (Callaini et al., 1991; Watts and Howard, 1992), there is no direct evidence that low temperature disrupts the actin cytoskeleton in plants. However, it should be noted that a wheat gene encoding an actin depolymerization factor (ADF/co®lin) is rapidly upregulated by cold (Danyluk et al., 1996). It is noteworthy, in this respect, that a recently characterized actin-defective yeast mutant is cold-sensitive (McCollum et al., 1999). Treatment of cells with 2 mM JK inhibits cold-induced Ca2+ in¯ux, cas30 expression and freezing tolerance substantially but not completely (Figure 3). JK concentrations from 0.1±10 mM had similar effects on cas30 expression (data not shown). This raises the possibility that these cold-induced processes may be at least partially modulated through, as yet unidenti®ed, mechanisms that operate independently of re-organization in the actin cytoskeleton. The substantial effects of CD and JK on Ca2+ in¯ux through opening of Ca2+ channels observed in this study indicate that such Ca2+ in¯ux requires the reorganization of the cytoskeleton. However, it should be emphasized that the cytoskeleton may also be a platform for several other physiological functions involved in cold acclimatization, such as protein traf®cking and modulation of activities of protein kinases/phosphatase that may or may not be dependent on Ca2+ in¯ux. An intriguing observation of the present study is that CD treatment at 25°C reduces the constitutive level of freezing ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 Early steps in cold sensing by plant cells 791 tolerance (Figure 5c) although it induces Ca2+ in¯ux and cas30 expression (Figure 5a,b). While the reasons for this are presently unclear, it is possible that triggering of CA requires only a transient disruption of the actin micro®lament network, and completion of the CA processes depends upon the re-organization of the actin cytoskeleton in an altered pattern. The required re-assembly of the actin cytoskeleton might be prevented by the continued presence of CD. The constitutive level of freezing tolerance is therefore lost by disruption of the pre-existing actin cytoskeleton coupled with a failure to re-organize. We are currently investigating the dynamics of actin cytoskeletal changes (F-actin levels) during cold shock as well as following treatments with modulators of membrane ¯uidity and actin micro®lament stabilization. What is the nature of mechanistic relationship between membrane rigidi®cation, actin micro®lament re-organization, and Ca2+ channel opening? The effects of CD and JK on Ca2+ in¯ux, observed in the present study, suggest that micro®laments are directly or indirectly connected to the channel, keeping it under tension in the closed state. It is tempting to suggest that cold- or chemically induced reorganization of actin micro®laments can dissipate the tension forces that keep the channel closed, thereby allowing the channel to open. This may explain why treatment with the actin micro®lament stabilizer JK would maintain the tension forces that keep the channel closed whereas CD could dissipate them. Our data suggest that re-organization of actin micro®laments in cold signalling is located downstream of membrane rigidi®cation, since the cas30 transcript accumulation induced by cold or by DMSO at 25°C is inhibited by JK (Figure 6c). Membrane rigidi®cation-induced re-organization of the actin micro®laments and Ca2+ channel opening would appear to require that the actin cytoskeleton be coupled to the plasma membrane as well as to Ca2+ channels. It is known that cytoskeletal components are attached to the plasma membrane and keep it under tension (Ding and Pickard, 1993; Schafer and Schroer, 1999). This observation assumes particular signi®cance because cold-activated Ca2+ channels in plants have been shown to be stretchactivated (Ding and Pickard, 1993), indicating that tensile forces are involved in the activation of these channels. In ®broblasts, controlled stretch forces applied to the plasma membrane cause large Ca2+ transients (Glogauer et al., 1995). It has also been shown that CD treatment induces Ca2+ transients through stretch-activated Ca2+ channels (Glogauer et al., 1995; Howarth et al., 1998; Wu et al., 1999; Xu et al., 1997). These Ca2+ transients do not occur following disruption of microtubules (Wu et al., 1999; Xu et al., 1997). Interestingly, Mazars et al. (1997) demonstrated that destabilization of microtubules with oryzalin increased Ca2+ in¯ux in cold-shocked tobacco protoplasts. We did not investigate the role of microtubules in lowã Blackwell Science Ltd, The Plant Journal, (2000), 23, 785±794 temperature signalling, but alfalfa suspension cells treated with oryzalin at 25°C do accumulate cas30 transcripts (OÈrvar et al., unpublished data), indicating that re-organization of both micro®laments and microtubules is involved in low-temperature sensing in these cells. The interaction between micro®laments and microtubules is well established and therefore it is possible that both these cytoskeletal components are involved in low-temperature signal transduction. To the best of our knowledge, this is the ®rst report demonstrating the signalling role of the actin cytoskeleton in regulating plant gene expression. It should be pointed out that the results presented here were derived from cell suspension cultures. Whether they can be extended to intact plants awaits further investigation. It has been proposed that the cytoskeleton acts as a scaffold where physical forces are transduced into biochemical signals (Schmidt and Hall, 1998). How temperature-induced changes in the physical state of the membrane regulate actin cytoskeleton organization remains to be determined. Cold-induced membrane rigidi®cation is believed to occur in distinct microdomains of the plasma membrane (Murata and Los, 1997). These microdomains may provide the loci within which low-temperature-induced membrane rigidi®cation modulates actin micro®lament organization. In various mammalian cell types, e.g. epithelial cells, Triton X-100-insoluble membrane microdomains called lipid rafts have been identi®ed (Anderson, 1998). These cholesterol-rich rafts move within the lipid bilayer and may serve as platforms for both transmembrane signal transduction and cytoskeletal re-arrangement (Anderson, 1998; Simons and Ikonen, 1997). Whether similar platforms or microdomains for transmembrane signalling and cytoskeletal remodelling exist in plants, and are important in cold sensing, awaits further investigation. Experimental procedures Plant material and cold acclimatization Conditions for growth of alfalfa (Medicago sativa ssp. falcata cv Anik) cell suspension cultures were as previously described (Monroy and Dhindsa, 1995). In all experiments, 7- or 8-day-old cultures were used. CA was administered by cooling the cells from 25 to 4°C (cooling rate 1.2°C min±1) under low light (20 mmol photons m±2 sec±1) and de-acclimatization by returning the cells to 25°C at 100 mmol photons m±2 sec±1 illumination. Administration of chemicals For modulation of membrane ¯uidity, benzyl alcohol (BA; Carratu et al., 1996; Kitagawa and Hirata, 1992) and DMSO (Lyman and Preisler, 1976) were added directly to the cell suspension at the concentrations indicated. The role of actin micro®lament reorganisation in the cold response was investigated using JK (Molecular Probes, Eugene, Oregon, USA) or CD (Sigma, Oakville, 792 BjoÈrn LaÂrus OÈrvar et al. Ontario, Canada). Stock solutions of both chemicals were made in 50% DMSO to ®nal concentrations of 0.7 and 3.94 mM, respectively. The ®nal treatment solutions of these chemicals contained 0.15% and 0.64% DMSO, respectively. To prevent Ca2+ in¯ux, the Ca2+ chelator 1,2-bis-(2-aminophenoxy)ethane N,N,N¢,N¢-tetraacetic acid (BAPTA; Sigma, Oakville, Ontario, Canada), dissolved in H2O, was used. For promoting Ca2+ in¯ux at 25°C, the Ca2+ agonist Bay K 8644 (Calbiochem, San Diego, California, USA) and the Ca2+ ionophore A23187 (ICN, Costa Mesa, California, USA), both dissolved in DMSO, were used as previously described (Monroy and Dhindsa, 1995). In each experiment, the cell suspension cultures were ®rst treated for 1 h with the indicated chemical at 25°C before incubation at 25°C or 4°C. In each experiment when relevant, control cultures were treated with the DMSO solvent concentration alone, as indicated in the ®gure legends. Cell viability and freezing tolerance Freezing tolerance of cell suspension cultures was determined by the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction method (Monroy et al., 1993) from two independent experiments and expressed as a percentage of viability of frozen, untreated control cells. RNA gel blot analysis Total RNA was isolated using TRIzol (Gibco BRL, Burlington, Ontario, Canada) according to the manufacturer's instructions, and 10 mg subjected to blot analysis with high-stringency hybridization and washing as described (Davis et al., 1994). For cas30 hybridization, the 5¢ end (480 bp BamHI/KpnI fragment) of cas30 corresponding to the previously reported cDNA clone pSM2358 (Mohapatra et al., 1989) was used as probe. To determine whether the chemical treatments investigated in this study affect cas30 speci®cally, the expression of two control genes was studied. A 250 bp Bsu36I/MunI cDNA fragment for the replacement histone H3.2 protein from alfalfa (Robertson et al., 1996), and a 721 bp cDNA fragment for a putative ribosomal protein L5 from alfalfa (Asemota et al., 1994), were used as probes to determine the transcript levels of their respective genes. All probes were radiolabelled with 32P by random priming (T7 QuickPrime Kit; Amersham Pharmacia, Baie d'UrfeÂ, QueÂbec, Canada) according to the manufacturer's instructions. Negative digital images of EtBr-stained blots were used as loading controls. Transcript levels following each treatment described were determined in at least three independent experiments. The results from one representative experiment are shown. Ca2+ in¯ux measurements Protoplasts, isolated from cell suspension cultures as described by Monroy and Dhindsa (1995) were used for Ca2+ in¯ux studies. The analysis of 45Ca2+ in¯ux by cation exchange chromatography was as previously described (Monroy and Dhindsa, 1995), except for the addition of 0.5 mM Ca2+ to stabilize membranes, and the use of 250±400 protoplasts per ml in each experiment. Protoplasts (200 ml) were ®rst treated for 30 min at 25°C with the chemical agents using gentle shaking (60 rev min±1), before incubation for 20 min (without shaking) with 45Ca2+ at 4°C (cooling rate 4°C min±1) or 25°C. Ca2+ in¯ux was compared to in¯ux in nontreated or DMSO-treated (solvent vehicle alone) protoplasts. Ca2+ in¯ux measurements were determined in at least three inde- pendent experiments, each with three replicates, using three different protoplast preparations. The results from one representative experiment are shown. Membrane ¯uidity measurements Membrane ¯uidity was determined using 1,6-diphenyl-1,3,5hexatriene (DPH, Sigma, Oakville, Ontario, Canada), prepared in azetonitrile, as probe. Protoplasts were treated at 25°C for 30 min, incubated with DPH (6 mM) for 30 min, followed by 20 min incubation at 4°C or 25°C. Steady-state ¯uorescence polarization measurements were carried out as described previously (Raymond and Plaa, 1996), using a Shimadzu RF-540 spectro¯uorophotometer with slit bandwidth of 10 nm and excitation and emission wavelengths of 365 and 432 nm, respectively. All experiments were repeated at least three times. The results from one representative experiment are shown and expressed as values of the ¯uorescence polarization index (p) that is inversely related to membrane ¯uidity (Choi and Yu, 1995). Protoplast viability The viability of treated protoplasts was determined by vital staining with Evan's blue (Kanai and Edwards, 1973). 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