Previous studies on cold-triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the β-glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25°C by the membrane rigidifier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25°C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B. Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP-ribose activated it at 25°C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25°C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25°C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold-activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases.
A rapid and transient rise in cytosolic Ca2+ level is known to be triggered by cold (Knight et al., 1991) and is required for cold-induced gene expression and cold acclimatization in alfalfa (Monroy et al., 1993b) and Arabidopsis (Tahtiharju et al., 1997). Most of the research on early events upstream of Ca2+ influx and in relation to cold acclimatization, has focused on alfalfa cell suspension cultures (Dhindsa et al., 1998) or on the unicellular cyanobacterium Synechocystis (Murata and Los, 1997; Nishida and Murata, 1996). Whether the results of these studies on unicellular systems are applicable to entire seedlings or their organs remains to be determined. Furthermore, previous studies have only monitored the accumulation of transcripts of end-point marker genes. However, the development of cold-induced freezing tolerance requires de novo protein synthesis (Chen et al., 1983). Whether the cold signaling cascade leads to the marker gene activity at the translational level is not known.
Here we examine the nature of early events leading to the cold activation of Brassica napus gene, BN115. We used a transgenic line of B. napus cv. Westar containing, in addition to the endogenous cold-inducible BN115 gene, the β-glucuronidase (GUS) gene placed under control of the BN115 promoter and thus rendered cold-inducible. Another transgenic line of the same cultivar contained GUS gene placed under the control of the tobacco cryptic constitutive promoter tCUP. Thus the seedlings of this line expressed GUS activity constitutively as a control for the cold-inducible BN115-driven GUS activity. As end-point markers of the cold signaling process, we monitored the accumulation of the endogenous BN115 transcripts, BN115-driven GUS activity, and development of freezing tolerance in Brassica napus leaves. The results of the present investigation show that in B. napus leaves (1) the cold activation of BN115 promoter is inhibited by the membrane fluidizer benzyl alcohol (BA), stabilizers of microfilaments and microtubules, Ca2+ chelators and channel blockers, and by inhibitors of several specific protein kinases, but is mimicked at 25°C by the membrane rigidifier DMSO, destabilizers of microfilaments and microtubules, Ca2+ ionophore A23187, cADP-ribose and inhibitors of protein phosphatases (PP) 1 and 2 A; and (2) activation of BN115 at 25°C by dimethylsulfoxide (DMSO), Ca2+ ionophore, cADPR or by inhibitors of protein phosphatases (PP) 1 and 2A, is accompanied by development of freezing tolerance.
Cold activation of the BN115 promoter-GUS activity in transgenic Brassica
The leaves of transgenic Brassica napus seedlings expressing GUS activity under the control of either the cold-inducible Brassica napus BN115 promoter, or the tobacco cryptic constitutive promoter tCUP, were exposed to 4°C for different times. The leaf discs were then punched out for determining GUS activity as a measure of BN115 activation. Leaves placed at 4°C exhibited high levels of BN115 promoter activity. The time required for the detection of activity varied with the level of expression examined, that is translational or transcriptional. When measured in terms of GUS activity, BN115 activation was hardly detectable at 24 h, but by 48 h it had reached maximum levels as indicated by the intensity of blue coloration of the leaf discs (Figure 1a). It should be noted that GUS activity expressed constitutively under the control of tCUP promoter was not affected by cold (Figure 1b). When BN115 activity was measured in terms of accumulation of its endogenous transcripts, it could be detected at high levels at 12 h of cold treatment (Figure 1c). The leaves kept at 25°C (0-time) did not show any activity (Figure 1a,c). To measure freezing tolerance, cold-acclimatized (CA) and nonacclimatized (NA) leaves were subjected to solute leakage test. It was found that solute leakage from NA leaves was more than twice that from CA leaves (Figure 1d), suggesting that CA leaves were more freezing tolerant than the NA leaves. It may therefore be concluded that BN115 promoter is able to confer cold-inducible expression on GUS activity, and that such activity may be used as a reliable marker to study the regulation of BN115 activation. Since both BN115 transcript levels and GUS activity were easily detected at 48 h of cold treatment, in all subsequent experiments this period of cold treatment was used when required.
Cold activation of BN115 requires membrane rigidification
Membrane fluidity is known to be directly and reversibly affected by temperature and cold-induced membrane rigidification is considered as the primary cold-sensing event (Murata and Los, 1997; Örvar et al., 2000; Vigh et al., 1993). Such membrane rigidification at low temperature can be prevented by chemical membrane fluidizers such as BA and mimicked at 25°C by treatment with the membrane rigidifier (DMSO) (Örvar et al. 2000) or by catalytic hydrogenation of membrane lipids (Vigh et al., 1993). Thus we examined the effects of BA and DMSO on BN115 activity. Leaves were treated with different concentrations of BA and exposed to 4°C. The data in Figure 1(e,f) show that increasing BA concentrations strongly inhibited the cold-inducible GUS activity (Figure 1e) but the constitutive GUS activity was not affected (Figure 1f). The cold-induced accumulation of BN115 transcripts was also inhibited by BA (Figure 1g) and the BA-treated leaves showed considerably more solute leakage than the untreated control leaves (Figure 1h). Thus treatment of leaves with BA inhibits the cold-induced BN115 activation and development of freezing tolerance.
Treatment of B. napus leaves with the membrane rigidifier DMSO at 25°C showed a marked increase in BN115-driven (Figure 1i), but not the constitutive (Figure 1j), GUS activity, and caused the accumulation of BN115 transcripts (Figure 1k). The solute leakage decreased with increasing concentration of DMSO (Figure 1l) suggesting that DMSO treatment results in an increased freezing tolerance at 25°C.
Cold activation of BN115 requires Ca2+ influx from cell wall and internal stores
Calcium is a well-known secondary messenger in many signaling pathways in plants, including temperature signaling (Trewavas and Mahlo, 1998). Since the cold-triggered Ca2+ influx may occur from Ca2+-rich cell wall and/or from intracellular Ca2+ stores, modulators of Ca2+ availability from both these sources were used in the present study. To examine the role of cell wall Ca2+, leaves were treated with a Ca2+ chelator EGTA or 1,2-bis(2-aminophenoxy)ethane N,N,N',N′-tetraacetic acid (BAPTA), or with a Ca2+ channel blocker La3+ or Gd3+, first at 25°C for 3 h and then at 4°C for 48 h. Treatment of leaves with each of these chemicals differentially decreased the cold-induced GUS activity (Figure 2a) but had no effect on the constitutive GUS activity (Figure 2b). These inhibitors of Ca2+ availability strongly inhibited the cold-induced accumulation of endogenous BN115 transcripts (Figure 2c). Gd3+, a specific blocker of mechanosensitive Ca2+ channels, was more effective than La3+ (a blocker of voltage-gated Ca2+ channels). The calcium inhibitors that reduced the cold-induced BN115 activity also decreased the cold-induced development of freezing tolerance as reflected by the increased solute leakage (Figure 2d). Treatment of leaves with ruthenium red (RuR), known to block Ca2+ release from internal stores, abolished the cold-induced, but not the constitutive, GUS activity (Figure 2a,b) and caused a large decrease in the development of freezing tolerance as shown by the increased solute leakage (Figure 2d). The effects of RuR were less pronounced on the cold-induced BN115 transcript accumulation (Figure 2c).
The effects of chemicals known to cause Ca2+ influx at 25°C were then examined. Treatment of leaves with the Ca2+ ionophore A23187, known to cause Ca2+ influx from the cell wall (Monroy and Dhindsa, 1995), or with cADPR which is known to release Ca2+ from internal stores (Allen et al., 1995), resulted in BN115 activation at 25°C. Thus A23187 or cADPR induced the BN115-driven (Figure 2e), but not the constitutive (Figure 2f) GUS activity. Both A23187 and cADPR caused the accumulation of BN115 transcripts at 25°C (Figure 2g) and increased the freezing tolerance as indicated by the reduced solute leakage from leaves (Figure 2h).
Recently, ruthenium red (RuR) has been shown to inhibit protein synthesis in monkey kidney cells (Creppy et al., 2000). Therefore, we examined the effects of RuR on the rate of protein synthesis in B. napus leaves. The effects of 0, 5, 10, and 20 µm RuR on the rate of incorporation of [35S]Methionine into TCA-precipitable fraction were determined. The data presented in Figure 3 show RuR at a concentration up to 20 µm had no effect on the rate of protein synthesis. In experiments reported in Figure 2(a)–(d,) 16.67 µm RuR was used. Therefore, at the concentration used in this study, RuR had no inhibitory effect on protein synthesis.
Cold activation of BN115 requires reorganization of cytoskeleton
Cold acclimatization has been shown to require rearrangements of cytoskeleton components, microtubules and actin microfilaments (Örvar et al., 2000). The chemicals that cause the rearrangements of these cytoskeletal structures also cause elevation in cytosolic Ca2+ levels (Mazars et al., 1997; Thion et al., 1996). We therefore investigated the role of the stability of microfilaments and microtubules in low temperature sensing leading to the activation of BN115. First, the effects of microfilament stabilizer jasplakinolide (JK) and the microtubule stabilizer taxol (Tax) were determined. Both JK and Tax inhibited the cold-induced (Figure 4a), but not the constitutive (Figure 4b) GUS activity. Each of these chemicals also inhibited the cold-induced accumulation of BN115 transcripts (Figure 4c) and caused increased solute leakage from the leaves (Figure 4d). Therefore, it may be concluded that stabilizers of microfilaments and microtubules prevent cold-induced BN115 activation and the development of freezing tolerance.
Next, the effects of microfilament destabilizer latrunculin B (LatB) and the microtubule destabilizers oryzalin (Ory) and colchicine (Col) were examined. We reasoned that if stabilizers of cytoskeleton prevent cold-induced activation of BN115 and development of freezing tolerance, then destabilization of the cytoskeleton may mimic the effects of cold at 25°C. Each of the three destabilizers used induced the BN115-driven GUS expression at 25°C (Figure 4e) but had no effect on the constitutively expressed GUS activity (Figure 4f). The accumulation of BN115 transcripts was also induced by each of the destabilizers at 25°C (Figure 4g). However, the cytoskeleton destabilizers had little effect on solute leakage (Figure 4h). Therefore, it may be concluded that destabilizers of microfilaments and microtubules cause the activation of BN115 but do not enhance freezing tolerance at 25°C.
Cold activation of BN115 involves action of several protein kinases and inhibition of protein phosphatases type 1 and 2A
Cold-induced gene expression and cold acclimatization have been shown to require rapid and reversible phosphorylation of specific pre-existing proteins (Monroy et al., 1993b). Therefore, we wished to determine if the cold activation of BN115 is affected by inhibitors of protein kinases and protein phosphatases. Results presented in Figure 5(a) show that general inhibitors of protein kinases, staurosporine and K252a, as well as the phosphoinositide kinase inhibitor wortmannin and the tyrosine kinase inhibitor genistein were each able to drastically reduce the cold-induction of GUS activity and BN115 transcript accumulation. Although not much is known about protein kinase C in plants, its inhibitor 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (H7) caused a strong reduction in the cold activation of BN115 promoter as measured by either GUS activity (Figure 5a) or BN115 transcript accumulation (Figure 5c). The inhibitor of cAMP-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H8) had little effect on the cold-induced activity of BN115. None of the protein kinase inhibitors had any effects on the constitutively expressed GUS activity (Figure 5b). Those protein kinase inhibitors that reduced the cold-activation of GUS activity and BN115 transcript accumulation, also reduced the development of freezing tolerance as shown by the increased solute leakage (Figure 5d). These observations suggest that activities of several types of protein kinases are required for the cold-induced activation of BN115 and development of freezing tolerance.
Next, the effects of inhibitors of protein phosphatases were examined. Since 85% of cellular protein phosphatase activity is attributed to protein phosphatases 1 and 2A, this study focused on the effects of inhibitors of these phosphatases. Okadaic acid (OA) or calyculin A (CalyA), each a potent inhibitor of PP1 and PP2A at low concentrations, had no effect on constitutively expressed GUS activity (Figure 5f) but was able to induce BN115-driven GUS activity (Figure 5e) as well as the accumulation of BN115 transcripts (Figure 5g) at 25°C. These protein phosphatase inhibitors also caused an increase in freezing tolerance as shown by the decrease in solute leakage (Figure 5h). It was therefore concluded that cold-induced activation of BN115 promoter and development of freezing tolerance may be associated with inactivation of PP1, PP2A or both.
We examined the nature of early events during cold signaling in B. napus seedlings by monitoring (1) the activity of BN115 promoter at the transcriptional and translational levels; and (2) the development of freezing tolerance in Brassica napus seedlings.
The direct and rapid effects of temperature on the physical state of biological membranes have been known for a long time (Levitt, 1980), and temperature-induced change in membrane fluidity has been considered a primary temperature sensing mechanism (Horvath et al., 1998; Murata and Los, 1997; Örvar et al. 2000). Thus, membrane rigidification by catalytic hydrogenation in Synechocystis (Vigh et al., 1993) and by DMSO in alfalfa cells (Örvar et al., 2000), results in the expression of cold-inducible genes at 25°C. The results of the present study show that cold activation of BN115 in Brassica napus leaves is prevented by membrane fluidizer benzyl alcohol and is mimicked at 25°C by the membrane rigidifier DMSO. The effects of cold, BA or DMSO are specific to the cold-inducible BN115 promoter as these treatments have little effect on the constitutive expression of GUS under the control of the tCUP promoter. It is noteworthy that DMSO treatment mimics the effects of cold completely including the development of freezing tolerance.
There is evidence in the literature that cold-induced gene expression and cold acclimatization in alfalfa (Monroy and Dhindsa, 1995; Monroy et al., 1993b) and Arabidopsis (Tahtiharju et al., 1997) requires Ca2+ influx into the cytosol. A temperature of 2°−5°C is routinely used in the laboratory to carry out cold acclimatization of plant cells and seedlings. Similar temperatures are known to activate mechanosensitive or stretch-activated Ca2+ channels (Ding and Pickard, 1993) and to cause maximum Ca2+ influx (Monroy and Dhindsa, 1995). The source of cold-induced Ca2+ influx has been shown to be largely cell wall in alfalfa (Monroy and Dhindsa, 1995) and Arabidopsis (Knight et al., 1996; Tahtiharju et al., 1997). An intriguing observation in the present study is that none of the two main sources of Ca2+ in plant cells, cell wall and the intracellular stores, by itself appears to be sufficient in causing the maximum activation of BN115. For example Gd3+, a specific blocker of mechanosensitive Ca2+ channels, caused a complete inhibition of cold-induced GUS activity as well as BN115 transcript accumulation (Figure 2a,b) while Ca2+ influx from intracellular stores was not blocked. This would suggest that the role of intracellular Ca2+ is insignificant. However, this notion is countered by the observed effects of RuR (Figure 2e,f), believed to block the release of Ca2+ from internal stores only (Allen et al., 1995). RuR completely abolished the cold-induced GUS activity and drastically reduced the accumulation of BN115 transcripts while Ca2+ entry from the cell wall was not blocked. Thus it appears that Ca2+ influx from both the cell wall and intracellular stores is required for low temperature signal transduction leading to BN115 activation. The combined contribution of these two sources of cold-triggered Ca2+ influx has not been observed before. However, it should be noted that previous studies either did not include gene expression at all (Knight et al., 1991), or monitored only the transcriptional level of gene expression (Knight et al., 1996; Monroy and Dhindsa, 1995; Tahtiharju et al., 1997). The present study examined the effects of Ca2+ availability on gene expression at the transcriptional and translational levels.
We have previously suggested (Monroy and Dhindsa, 1995) that the cold-induced membrane rigidification may be coupled to the opening of mechanosensitive Ca2+ channels. However, the nature of the coupling processes is unclear. Using alfalfa cells, we have recently provided evidence that cytoskeleton rearrangements may mediate the transduction of the cold signal from the rigidified membrane to the Ca2+ channels (Örvar et al., 2000). Remodeling of cytoskeleton is known to mediate cell responses to a variety of signals (Mathur et al., 1999) and it has been suggested that cytoskeleton acts as a scaffold to the changes which transduce physical forces into biochemical signals (Schmidt and Hall, 1998). This suggestion assumes particular significance because cold activated Ca2+ channels have been shown to be mechanosensitive in nature (Ding and Pickard, 1993) and because Gd3+, a blocker of mechanosensitive Ca2+ channels, prevents the cold activation of BN115 almost completely (Figure 2a,c). Changes in membrane fluidity are likely to alter tensile forces operating in the membrane. It is well established that cytoskeleton components are attached to the plasma membrane and ion channels (Trewavas and Malho, 1998). If cytoskeleton remodeling is required for cold signaling, then the stabilizers of microfilaments and microtubules are expected to prevent it whereas their destabilizers/depolymerizers should initiate it. The results of the present study show that treatment of leaves with either the microtubule stabilizer taxol or the microfilament stabilizer jasplakinolide strongly inhibits the cold activation of BN115 promoter. On the other hand, the microtubule destabilizer oryzalin or colchicine, or the microfilament destabilizer latrunculin B, activates the BN115 promoter at 25°C. This suggests that cold activation of BN115 requires reorganization of both microtubules and microfilaments. Destabilization of microfilaments and microtubules causes Ca2+ influx in plant cells (Mazars et al., 1997; Thion et al., 1996), whereas a stabilization of the actin microfilaments inhibits the cold-induced Ca2+ influx, gene expression and development of freezing tolerance in alfalfa cells (Örvar et al., 2000).
Cold- or chemically-induced BN115-driven expression of GUS activity demonstrates the regulation of BN115 at the translational level. The effects of chemicals that prevent cold activation of BN115 (such as membrane fluidizers, cytoskeleton stabilizers and Ca2+ chelators and channel blockers) are consistently more pronounced on the GUS activity than on the level of BN115 transcripts. This is especially true of the effects of Ruthenium red (RuR, Figure 2a,c). Our data show that the observed effects of RuR are specifically on the cold-inducible BN115 activity because RuR has no effect on the constitutively expressed GUS activity under the control of tCUP promoter (Figure 1b) and, at the concentrations used, RuR had no effect on protein synthesis (Figure 3).
The effects of several chemicals that mimic the effects of cold and activate BN115 at 25°C are more pronounced on GUS activity than on the level of BN115 transcripts, although both genes are transcribed under control of the same promoter. This may reflect possible differences between the stability of mRNAs of the two genes. The transcripts of cold-inducible genes are known to be extremely unstable at 25°C. For example, the half life of cas18 transcripts in alfalfa is greater than 100 h at 4°C but less than 30 min at 25°C (Wolfraim et al., 1993). Since GUS gene under its own promoter is not cold inducible, its transcripts, even when made under the control of BN115 promoter, may be much more stable than the endogenous BN115 transcripts at 25°C. Thus the chemical induction of BN115 at 25°C is seen to be much lower in terms of BN115 transcript accumulation than in terms of GUS activity. Cold- or chemically-induced GUS activity demonstrates that the regulation of BN115 by these treatments is manifested at the translational level. However, our data do not rule out the possibility that the same treatments may regulate the endogenous BN115 protein levels differently.
Mediation of cold acclimatization by protein phosphorylation has been demonstrated (Dhindsa et al., 1998; Monroy et al., 1993b; Tahtiharju et al., 1997) and evidence for the differential roles of protein kinases and protein phosphatases in cold-induced gene expression has been reported (Monroy et al., 1997, 1998). In addition to the general inhibitors of protein kinases, staurosporine and K252a, the specific inhibitors of tyrosine kinases (genistein), phosphoinositide kinases (wortmannin), and of protein kinase C (H7), all strongly inhibit the cold activation of BN115 (Figure 4). The strongest inhibition is by genistein, wortmannin and H7. These data suggest that cold signaling, as expected, is a complex multistep process and involves the role of several types of protein kinases. The inhibition of cold activation of BN115 by the protein kinase C (PKC) inhibitor H7 is interesting because the structural and catalytic features and role of this enzyme in plants are still unclear.
It should be noted that all chemical treatments that mimic the effects of cold in activating the BN115 promoter at 25°C, except the destabilizers of cytoskeleton, also confer freezing tolerance. Thus they mimic all the cold-triggered events leading up to and including the development of freezing tolerance. The destabilizers of microfilaments (LatB) and microtubules (oryzalin and colchicine) do not cause any increase in freezing tolerance (or reduce solute leakage, Figure 4h), although they activate the BN115 promoter. While the reasons for this are presently unclear, it is likely that the continued presence of these chemicals prevents the repolymerization of the cytoskeleton in an altered pattern essential for cold acclimatization. Another microfilament destabilizer cytochalasin D had similar effects in alfalfa cells (Örvar et al., 2000).
In conclusion, we have investigated the nature of events involved in the cold activation of BN115 promoter in Brassica napus leaves. As end-point markers, we used the accumulation of BN115 transcripts, BN115-driven GUS activity, and development of freezing tolerance. The results show that cold activation of BN115 is mediated by membrane rigidification, cytoskeleton rearrangements and Ca2+ influx and involves the role of several types of protein kinases. Using alfalfa cell suspensions, we have recently provided evidence that cold signal is transmitted from the rigidified membrane to the Ca2+ channels via rearrangements in cytoskeleton (Örvar et al., 2000). The challenge now is to identify the nature of cold-triggered rearrangements in cytoskeleton and mechanisms leading to the opening of Ca2+ channels.
Plant material and cold acclimation
Three-w-old transgenic seedlings of Brassica napus cv. Westar were used in all experiments. In addition to the endogenous cold-inducible BN115 gene, the seedlings also contained the coding sequence of β-glucuronidase (GUS) reporter gene rendered cold inducible by its fusion to the BN115 promoter (White et al., 1994). Another transgenic line of B. napus cv. Westar expressed GUS activity constitutively. It carried the GUS coding sequence under control of the tobacco cryptic constitutive promoter tCUP (Foster et al., 1999). The seedlings of this line were used as control constitutive GUS expression as opposed to the cold-inducible BN115-driven GUS expression. In all experiments, constitutive GUS expression refers to the tCUP-driven GUS expression. All other determinations were made on seedlings carrying BN115-GUS fusion. All plants were grown at 20°C under a 16-h photoperiod and 200 µmol m−2s−1 light intensity. Cold treatment was administered by placing leaves at 4°C under low light intensity (20 µmol m−2s−1).
In order to administer chemical treatments, leaves of equal surface area were cut diagonally at the base of the petiole with a sharp razor and placed in half strength Hoagland's solution containing 0.02% Tween-20, with or without the treatment chemical. Thus chemicals were administered to the leaves via the transpiration stream through the petioles. Leaves to be exposed to cold were treated with the respective chemical first at 25°C for 3 h and then the treatment was continued at 4°C for times indicated in the respective figure legends. Leaves to be maintained at 25°C were treated with the chemical at 25°C for times indicated. Harvesting involved cutting the leaf blade at the mid-rib and immediately freezing one half of the leaf in liquid nitrogen for RNA extractions. Leaf discs were punched out from the other half of the leaf and used to perform GUS assays. BN115 is not induced by wounding (Weretilnyk et al., 1993). In all experiments where a solvent other than water was used to dissolve the treatment chemical, control incubation medium (C) contained the same solvent concentration as the treatment medium.
Concentrations of different chemicals used in various experiments are given in respective figure legends.
Histochemical assay of GUS activity
At termination of the experiment, leaf discs were punched out and washed in 90% acetone for 15 min and then rinsed for 5 min in rinse solution (50 mm sodium phosphate, pH 7.2, 0.5 mm K3Fe(CN)6, 0.5 mm K4Fe(CN)6). The discs were then placed for 24–48 h in rinse solution containing 1.5 mm 5-bromo-4-chloro-3-indoyl glucuronide (X-Gluc, Rose Scientific, Edmonton, Alberta, Canada) and 0.05% Triton-X-100. Chlorophyll was removed by washing discs in 30% ethanol for 1 h, and in 50% FAA (50% ethanol, 5% acetic acid, 3.7% formaldehyde) for 1 h. Discs were then stored in 70% ethanol until photographed.
Extraction of total RNA
Leaves were ground in liquid nitrogen and RNA was extracted using TRIzol (Gibco BRL, Burlington, Ontario, Canada), following the manufacturer's instructions.
Preparation of cDNA probe
A plasmid containing BN115 cDNA, pBN115, was cut with EcoRI and the released 800-bp fragment, corresponding to the BN115 cDNA, was gel-purified and radiolabeled by random priming using the T7 QuickPrime® Kit (Pharmacia; Amersham Pharmacia, Baie d'Urfé, Québec, Canada), and [α-32P]dCTP, following the manufacturer's instructions. The constitutively expressed p2.1 was used as a control gene as previously described (Monroy et al., 1993a).
RNA gel-blot analysis
Total RNA was separated on a 1.5% formaldehyde gel, transferred to a nylon membrane (Biotrans, ICN, Costa Mesa, California, USA) overnight, UV cross-linked, and the membrane was dried. Blots were hybridized overnight (7% SDS, 0.25 m phosphate buffer, pH 7.4, 2 mm EDTA) with the probe in a Robbins scientific hybridization incubator, Model 400, at 60°C and 15 r.p.m. Washes, 15 min each at 60°C, were twice in 1X SSC, 0.1% SDS, and once in 0.1X SSC, 0.1% SDS.
Determination of solute leakage
Solute leakage was used as an inverse measure of freezing tolerance. Treated leaves were rolled and placed in Falcon 2059 tubes in a freezing water bath kept at 0°C for 30 min. Samples were then seeded with a small piece of ice and kept at 0°C for another 60 min, after which the temperature was decreased at a rate of 2°C/h to −4°C, followed by −5°C for 30 min. Samples were then placed at 4°C overnight. Water was added to the tubes and electrolyte leakage measured after 4 h of gentle shaking at 25°C (Reading 1). Samples were placed at −80°C overnight, and the electrolyte leakage measured after shaking at 25°C for 4 h (Reading 2). Percent solute leakage was calculated by dividing reading 1 by reading 2 and multiplying the result by 100. Cold acclimatization for determining solute leakage experiments was carried out for 6 d at 4°C.
Determination of the rate of protein synthesis
Leaf discs were treated with varying concentrations of ruthenium red in half-strength Hoagland solution for 3 h at 25°C, leaf discs were punched out placed in half-strength Hoagland solution containing 50µCi [35S]methionine (1000 Ci mmol−1, Amersham Pharmacia) for 3 h. The leaf discs were then washed with half strength Hoagland's solution containing 1 mg l−1 methionine, ground to a powder in liquid nitrogen and protein extract prepared by adding 2 volumes (w/v) of buffer (100 mm HEPES, pH 7.5, 5 mm EDTA, 5 mm EGTA, 10 mm DTT, 10 mm Na3VO4, 10 mm NaF, 50 mmβ-mercaptoethanol, 1 mm phenlymethylsulfonyl flouride, 5 µg ml-1 antipain, 5 µg ml-1 aprotinin, 5 µg ml-1 leupeptin, 10% glycerol, 7.5% polyvinylpolypyrrolidone). The homogenate was centrifuged for 20 min at 13 000 r.p.m. Supernatant was transferred to another tube, and protein amount was assayed using the Bradford assay (BioRad). An identical aliquot of the extract was used to precipitate proteins with 5% TCA. The radioactivity in the aqueous suspension of the proteins was determined by scintillation counting. The results presented are as means of 3 replicates with error bars representing standard deviation.
All experiments were repeated at least 3 times and yielded similar results each time.
Work supported by Grant A2724 from the Natural Sciences and Engineering Research Council of Canada. We thank Dow AgroSciences (Indiana, USA) for providing oryzalin, and Dr B. Miki, Agriculture and Agri-Food Canada, Eastern Cereal Research Centre, Ottawa, Canada for providing seeds of transgenic B. napus line expressing GUS activity under control of the tobacco cryptic constitutive promoter tCUP.