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Keywords:

  • cold acclimatization;
  • signalling;
  • actin;
  • membrane fluidity

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 influx into the cytosol. Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold-acclimatize. Conversely, chemical agents causing increased calcium influx induce cold acclimatization-specific (cas) gene expression in alfalfa at 25°C. How low temperature triggers calcium influx is, however, unknown. We report here that induction of a CA-specific gene (cas30), calcium influx and freezing tolerance at 4°C are all prevented by cell membrane fluidization, but, conversely, are induced at 25°C by membrane rigidification. cas30 expression and calcium influx at 4°C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25°C by the actin microfilament destabilizer cytochalasin D (CD). JK blocked the membrane rigidifier-induced, but not the calcium channel agonist-induced cas30 expression at 25°C. These findings 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 rigidification and calcium influx in CA.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 influx of Ca2+ into the cytosol is an early event during CA ( Knight et al. 1991 ; Monroy & Dhindsa, 1995). Blocking this Ca2+ influx 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+ influx, such as the Ca2+ channel agonist Bay K 8644 or the Ca2+ ionophore A23187, induce CA-specific (cas) gene expression in alfalfa cells at 25°C ( Monroy & Dhindsa, 1995). These results demonstrate that cold-induced influx 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+ influx are largely unknown.

A direct and perhaps the earliest effect of an alteration of temperature on cells is the change in fluidity of their membranes ( Levitt, 1980). A decrease in temperature lowers membrane fluidity, whereas its increase enhances membrane fluidity ( Alonso et al. 1997 ; Mejia et al. 1995 ). Previous studies ( Murata & Los, 1997) have suggested that the plasma membrane acts as the primary sensor of temperature change through dynamic changes in its physical state. Rigidification 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 fluidity modulates ion channel activity. Mazars et al. (1997) have shown that cold-induced Ca2+ influx in tobacco protoplasts was stimulated by disruption of microtubules and actin microfilaments. Recent studies have also demonstrated roles for actin microfilaments 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 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 fluidity and re-organization of the actin cytoskeleton have a role in the early stages of low-temperature signalling in plant cells. We also investigated whether these events are linked to Ca2+ influx and CA. We have used the transcript level of a CA-specific 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 rigidification through actin microfilament re-organization to Ca2+ influx and CA-specific gene expression, eventually leading to CA and increased frost tolerance.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Chemical modulation of membrane fluidity in alfalfa protoplasts

Both DMSO and benzyl alcohol (BA) have been routinely used as a membrane rigidifier and fluidizer, respectively. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to confirm the effects of these agents on alfalfa protoplasts, but the polarization index p is inversely related to membrane fluidity ( Choi & Yu, 1995). The data presented in Table 1 show that DMSO treatment reduces membrane fluidity 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 fluidity.

Table 1.  Polarization index (p) of isolated protoplasts treated with cold, BA, DMSO, JK or CD
TreatmentTemperature (°C)Polarization index (p)
  1. Data are expressed as the mean ± SD of three replicates. Values with different letters are significantly different (P < 0.05) in each experiment as determined by Student's t-test.

Experiment 1
Control250.0954 ± 0.0073a
Control40.1315 ± 0.0138b
Experiment 2
Control250.0825 ± 0.0043a
Control40.1173 ± 0.0122b
BA (5 m m) 40.0853 ± 0.0064a
Experiment 3
Control250.0870 ± 0.0015a
Control40.1322 ± 0.0048b
DMSO (3%)250.1003 ± 0.0029c
Experiment 4
Control250.0921 ± 0.0109a
Control40.1555 ± 0.0136b
JK (2 μm) 40.1525 ± 0.0085b
Experiment 5
Control250.1084 ± 0.0115a
CD (50 μm) 250.1053 ± 0.0102a

Membrane fluidization inhibits cold signalling in alfalfa cells

To test the hypothesis that membrane fluidity plays a fundamental role in low-temperature signal transduction in alfalfa, transcript accumulation of a CA-specific gene, cas30, and the development of freezing tolerance in alfalfa cell suspension cultures were used as end-point markers. The expression of cas30 is specifically 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 ( Fig. 1; data not shown). BA, a well-established membrane fluidizer ( Carratu et al. 1996 ; Kitagawa & Hirata, 1992), was used to counter cold-induced membrane rigidification. 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 ( Fig. 2a). BA treatment also strongly inhibited the development of freezing tolerance ( Fig. 2b) without affecting cell viability (data not shown). Similar results were obtained by treating cells with ethanol, another membrane fluidizer ( 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 de-acclimatization ( Fig. 1), are also not affected by BA treatment ( Fig. 2a). Thus the effects of BA on cas30 transcript levels do not reflect a general down-regulation of transcription.

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Figure 1. Cas30 transcript accumulation during 48 h cold acclimatization (CA), and subsequent disappearance following de-acclimatization (DA) at 25°C.

Total RNA (10 μg) 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 confirmed by negative imaging of the RNA blot.

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image

Figure 2. Membrane fluidization inhibits cold-triggered cas30 transcript accumulation, development of freezing tolerance and Ca2+ influx in alfalfa cells.

(a) Inhibition of cold-induced cas30 transcript accumulation by the membrane fluidizer 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 cold-triggered Ca2+ influx. Protoplasts were treated with 5 m m BA for 30 min, followed by a 20 min incubation with 45Ca2+ at 4°C. 45Ca2+ influx is expressed as the ratio (%) of BA-treated versus untreated (C) protoplasts, both at 4°C. Although 20 m m BA treatment did not affect the viability of intact cells, it reduced the viability of isolated protoplasts by 50%, whereas 5 m m 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.

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CA in alfalfa suspension cells involves a rapid influx of Ca2+ into the cytosol as determined by 45Ca2+ cation exchange chromatography as previously described ( Monroy & Dhindsa, 1995). Isolated protoplasts were used to measure Ca2+ influx 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 cold-induced Ca2+ influx has been shown to be primarily apoplastic in alfalfa cells and is strongly inhibited by Ca2+ chelators and Ca2+ channel blockers ( Monroy & Dhindsa, 1995). Using 45Ca2+ cation exchange chromatography ( Monroy & Dhindsa, 1995), we examined the effects of 5 m m BA treatment on cold-induced Ca2+ influx. 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 rigidification ( Table 1). Treatment of protoplasts with 5 m m BA inhibited Ca2+ influx ( Fig. 2c). Thus, at 4°C, Ca2+ influx, cas30 gene expression and freezing tolerance are all prevented by BA-induced cell membrane fluidization.

Membrane rigidification activates cold signalling in alfalfa cells at 25°C

We reasoned that if reduced membrane fluidity mediates the cold-induced cas30 transcript accumulation, Ca2+ influx and increased freezing tolerance, then reduction of membrane fluidity at 25°C by chemical agents should mimic these responses. Treatment of cells at 25°C with DMSO, a well-documented membrane rigidifier ( Lyman & Preisler, 1976), at concentrations as low as 1%, induced cas30 transcript accumulation ( Fig. 3a), but did not affect the H3.2 and L5 mRNA levels ( Fig. 3a). DMSO treatment at 25°C increased membrane rigidification in isolated protoplasts ( Table 1), enhanced the freezing tolerance of cells ( Fig. 3b) and promoted Ca2+ influx ( Fig. 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+ influx and increased freezing tolerance, can be induced by membrane rigidification at 25°C, or prevented at 4°C by membrane fluidization.

image

Figure 3. Membrane rigidification induces cas30 transcript accumulation, development of freezing tolerance and Ca2+ influx in alfalfa cells at 25°C.

(a) Induction of cas30 transcript accumulation by the membrane rigidifier 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+ influx by DMSO at 25°C, compared to cold-induced Ca2+ influx. 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.

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Disruption of actin microfilaments is required for cold signalling in alfalfa cells

Yamamoto 1989) demonstrated that membrane rigidifier-induced Ca2+ influx in hepatocytes is accompanied by actin microfilament re-organization. It has also been shown that cold-induced Ca2+ influx in tobacco protoplasts can be enhanced (almost doubled) by chemical such as cytochalasin D (CD) that modulate actin microfilament organization ( Mazars et al. 1997 ). We therefore examined whether modulators of actin microfilament re-organization mimic the effect of cold-induced rigidification of the membrane on Ca2+ channels and cas30 expression. To modulate actin microfilament 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 plant actin microfilaments. Treatment with JK inhibits cold-induced cas30 transcript accumulation ( Fig. 4a) and Ca2+ influx ( Fig. 4b), and lowers freezing tolerance ( Fig. 4c). Conversely, treatment of cells with CD caused both cas30 transcript accumulation ( Fig. 5a) and increased Ca2+ influx ( Fig. 5b) at 25°C. However, CD treatment at 25°C reduced the constitutive level of freezing tolerance ( Fig. 5c). Cell or protoplast viability was unaffected by treatment with JK or CD (data not shown). Furthermore, membrane fluidity ( Table 1) and transcript levels of H3.2 and L5 were apparently unaffected by either JK or CD treatment ( Fig. 4a and Fig. 5a). Thus, Ca2+ influx and cas30 expression are induced by an actin microfilament destabilizing agent, and, conversely, inhibited by an actin microfilament stabilizing agent.

image

Figure 4. Cold-induction of cas30 transcript accumulation, Ca2+ influx and freezing tolerance are inhibited by the actin microfilament 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 μm) 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+ influx by JK. Protoplasts were treated for 30 min with JK (2 μm) or solvent vehicle alone (C) at 25°C before incubation with 45Ca2+ at 4°C for 20 min. (c) Inhibition of cold-induced development of freezing tolerance by JK. Cells were treated for 1 h at 25°C with JK (2 μm) 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.

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image

Figure 5. cas30 transcripts accumulation and Ca2+ influx are induced by the actin microfilament 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 μm) or solvent vehicle alone (C; 0.64% DMSO). (b) Induction of Ca2+ influx by CD. Protoplasts were treated for 30 min with CD (50 μm) 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 μm) 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.

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Temporal order of membrane rigidification and actin microfilament re-arrangement in cold signalling in alfalfa cells

The results presented above suggest that cold-induced rigidification of membranes and actin microfilament re-organization are required for Ca2+ influx 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+ influx ( Monroy et al. 1993 ). The data in Fig. 6 show that cas30 expression induced at 25°C by either DMSO treatment (membrane rigidifier) or by CD treatment (microfilament destabilizer) is inhibited by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid (BAPTA, Fig. 6a,b). These results indicate that Ca2+ influx is downstream of both membrane rigidification and actin microfilament re-organization, and further extend the results presented in Fig. 3(c) and 5(b). The DMSO-induced cas30 expression at 25°C is also inhibited by the actin microfilament stabilizer, JK ( Fig. 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 ( Fig. 6d). These data demonstrate that cold-induced re-organization of actin microfilaments is located downstream of membrane rigidification but upstream of Ca2+ influx.

image

Figure 6. Cold-induced re-organization of actin microfilaments provides a link between membrane rigidification and Ca2+ influx.

(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 m m) 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 m m) and then for 3 h with CD (50 μm). Control cells were treated with CD (50 μm) 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 μm) 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 μm) or BAPTA (2 m m) and then for 3 h with Bay K 8644 (BK; 100 μm). Control cells were treated with Bay K 8644 (100 μm) or solvent vehicle alone (C) alone for 3 h.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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+ influx, cas30 transcript accumulation and development of freezing tolerance are inhibited by chemicals that either fluidize membranes or stabilize actin microfilaments; (b) chemicals that either rigidify membranes or destabilize actin microfilaments cause Ca2+ influx and cas30 expression at 25°C; and (c) the role of actin cytoskeleton in low-temperature signal transduction is located downstream of membrane rigidification but upstream of Ca2+ influx.

The importance of membrane fluidity in low-temperature perception has long been recognized ( Levitt, 1980; Murata & Los, 1997). However, its relationship to subsequent responses to cold has been unclear. Our results show that DMSO treatment induces membrane rigidification and mimics the effects of cold on gene expression, Ca2+ influx and cold acclimatization. Furthermore, counteracting the cold-induced membrane rigidification with chemical fluidizers halts all examined responses to cold, suggesting that membrane rigidification is not only a sufficient 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 rigidification.

It is widely accepted that Ca2+ influx into the cytosol is triggered by cold ( Knight et al. 1991 ) and is required for cold-induced gene expression and development of freezing tolerance ( Knight et al. 1996 ; Monroy & Dhindsa, 1995; Monroy et al. 1993 ; Tahtiharju et al. 1997 ). The data presented here indicate that cold-triggered Ca2+ influx is downstream of membrane rigidification, because the influx is inhibited by membrane fluidization ( Fig. 2c) but triggered at 25°C by membrane rigidification ( Fig. 3c). Although membrane rigidification-induced Ca2+ influx has not been reported in plants, DMSO-induced membrane rigidification induces Ca2+ influx in hepatocytes ( Yamamoto, 1989) and parathyroid cells ( Nygren et al. 1987 ). Cholesterol, another frequently used modulator of membrane fluidity, increases membrane rigidity and causes Ca2+ influx in rabbit smooth muscle cells ( Gleason et al. 1991 ). Thus, membrane rigidification and Ca2+ influx 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 influx 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+ influx ( Fig. 5b) and cas30 expression at 25°C ( Fig. 5a). Conversely, treatment with JK inhibits both these events at 4°C ( Fig. 3a,b). JK can simultaneously stabilize one population of actin microfilaments 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 (Örvar et al. unpublished data), indicating that JK stabilizes the microfilaments. However, the mechanism of CD action is quite complex. CD can cap the barbed end of actin microfilaments, severe the microfilaments and sequester actin monomers, and thus affect the abundance and organization of microfilaments ( Cooper, 1987; Goddette & Frieden, 1986; Sampath & Pollard, 1991). Although latrunculin B, a powerful actin microfilament-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 ( Fig. 5b), does not require depolymerizaton of microfilaments, but instead remodelling of the pre-existing network. To date, although low-temperature disruption of the actin cytoskeleton has been demonstrated in animal cells ( Callaini et al. 1991 ; Watts & 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/cofilin) is rapidly up-regulated 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 μm JK inhibits cold-induced Ca2+ influx, cas30 expression and freezing tolerance substantially but not completely ( Fig. 3). JK concentrations from 0.1–10 μm 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 unidentified, mechanisms that operate independently of re-organization in the actin cytoskeleton. The substantial effects of CD and JK on Ca2+ influx through opening of Ca2+ channels observed in this study indicate that such Ca2+ influx requires the re-organization 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 trafficking and modulation of activities of protein kinases/phosphatase that may or may not be dependent on Ca2+ influx.

An intriguing observation of the present study is that CD treatment at 25°C reduces the constitutive level of freezing tolerance ( Fig. 5c) although it induces Ca2+ influx and cas30 expression ( Fig. 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 microfilament 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 fluidity and actin microfilament stabilization.

What is the nature of mechanistic relationship between membrane rigidification, actin microfilament re-organization, and Ca2+ channel opening? The effects of CD and JK on Ca2+ influx, observed in the present study, suggest that microfilaments 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 re-organization of actin microfilaments can dissipate the tension forces that keep the channel closed, thereby allowing the channel to open. This may explain why treatment with the actin microfilament 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 microfilaments in cold signalling is located downstream of membrane rigidification, since the cas30 transcript accumulation induced by cold or by DMSO at 25°C is inhibited by JK ( Fig. 6c). Membrane rigidification-induced re-organization of the actin microfilaments 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 & Pickard, 1993; Schafer & Schroer, 1999). This observation assumes particular significance because cold-activated Ca2+ channels in plants have been shown to be stretch-activated ( Ding & Pickard, 1993), indicating that tensile forces are involved in the activation of these channels. In fibroblasts, 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+ influx in cold-shocked tobacco protoplasts. We did not investigate the role of microtubules in low-temperature signalling, but alfalfa suspension cells treated with oryzalin at 25°C do accumulate cas30 transcripts (Örvar et al. unpublished data), indicating that re-organization of both microfilaments and microtubules is involved in low-temperature sensing in these cells. The interaction between microfilaments 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 first 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 & Hall, 1998). How temperature-induced changes in the physical state of the membrane regulate actin cytoskeleton organization remains to be determined. Cold-induced membrane rigidification is believed to occur in distinct microdomains of the plasma membrane ( Murata & Los, 1997). These microdomains may provide the loci within which low-temperature-induced membrane rigidification modulates actin microfilament organization. In various mammalian cell types, e.g. epithelial cells, Triton X-100-insoluble membrane microdomains called lipid rafts have been identified ( 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 & 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

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material and cold acclimatization

Conditions for growth of alfalfa (Medicago sativa ssp. falcata cv Anik) cell suspension cultures were as previously described ( Monroy & 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 μmol photons m−2 sec−1) and de-acclimatization by returning the cells to 25°C at 100 μmol photons m−2 sec−1 illumination.

Administration of chemicals

For modulation of membrane fluidity, benzyl alcohol (BA, Carratu et al. 1996 ; Kitagawa & Hirata, 1992) and DMSO ( Lyman & Preisler, 1976) were added directly to the cell suspension at the concentrations indicated. The role of actin microfilament reorganisation in the cold response was investigated using JK (Molecular Probes, Eugene, Oregon, USA) or CD (Sigma, Oakville, Ontario, Canada). Stock solutions of both chemicals were made in 50% DMSO to final concentrations of 0.7 and 3.94 m m, respectively. The final treatment solutions of these chemicals contained 0.15% and 0.64% DMSO, respectively. To prevent Ca2+ influx, 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+ influx 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 & Dhindsa, 1995). In each experiment, the cell suspension cultures were first 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 figure 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 μg 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 specifically, 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'Urfé, Québec, 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+ influx measurements

Protoplasts, isolated from cell suspension cultures as described by Monroy & Dhindsa 1995) were used for Ca2+ influx studies. The analysis of 45Ca2+ influx by cation exchange chromatography was as previously described ( Monroy & Dhindsa, 1995), except for the addition of 0.5 m m Ca2+ to stabilize membranes, and the use of 250–400 protoplasts per μl in each experiment. Protoplasts (200 μl) were first 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+ influx was compared to influx in non-treated or DMSO-treated (solvent vehicle alone) protoplasts. Ca2+ influx measurements were determined in at least three independent experiments, each with three replicates, using three different protoplast preparations. The results from one representative experiment are shown.

Membrane fluidity measurements

Membrane fluidity was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH, Sigma, Oakville, Ontario, Canada), prepared in azetonitrile, as probe. Protoplasts were treated at 25°C for 30 min, incubated with DPH (6 μm) for 30 min, followed by 20 min incubation at 4°C or 25°C. Steady-state fluorescence polarization measurements were carried out as described previously ( Raymond & Plaa, 1996), using a Shimadzu RF-540 spectrofluorophotometer 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 fluorescence polarization index (p) that is inversely related to membrane fluidity ( Choi & Yu, 1995).

Protoplast viability

The viability of treated protoplasts was determined by vital staining with Evan's blue ( Kanai & Edwards, 1973). Briefly, protoplasts where treated with BA (5 or 20 m m), DMSO (3%), JK (2 μm), CD (50 μm) or solvent alone, for 30 min at 25°C, with gentle shaking (60 rev min−1), before incubation at 4°C or 25°C. The number of protoplasts were counted in 16 squares in a haemocytometer and the average viability ± SD of eight replicates calculated.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Drs B. Tuchweber and M. Audet for their valuable help with the membrane fluidity measurements, Dr R. Esnault for the L5 cDNA, Drs T. Kapros and J.H. Waterborg for the H3.2 cDNA, and Drs T. Bureau, B. Suter, Ronald Poole and U. Thorsteinsdottir for the critical reading of the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada.

References

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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