Pharmacological Evidence for Calcium Involvement in the Long-Term Processing of Abiotic Stimuli in Plants

Abstract

Information about abiotic conditions is stored for long periods in plants and, in flax seedlings, can lead to the production of meristems. To investigate the underlying mechanism, flax seedlings were given abiotic stimuli that included a mechanical stimulus (by manipulation), one or two cold shocks, a slow cold treatment and a drought stress and, if these seedlings were then subjected to a temporary (1 to 3 days) depletion of calcium, epidermal meristems were produced in the seedling hypocotyls. This production was inhibited by the addition to the nutrient media of EGTA, ruthenium red, lanthanum or gadolinium that affect calcium availability or calcium transport. Use of these agents revealed a period of vulnerability in information processing that was less than two min for mechanical stimuli and over five min for other abiotic stimuli, consistent with information about mechanical stimuli being stored particularly fast. We propose that external calcium is needed for the transduction/storage of the information for meristem production whilst a temporary depletion of external calcium is needed for the actual production of meristems. Such roles for calcium would be consistent with a mechanism based on ion condensation on charged polymers.

Introduction

Plants are sensitive to many different abiotic stimuli from their environment (such as rain, wind, touch, drought or thermal stresses), to which they usually respond by adaptive modifications of their development.¹ To achieve such adaptation, plants must obtain and exploit a representative sample of the different factors characteristic of their environment. We have proposed that this entails seedlings registering and storing information from different sources over several weeks or months and then integrating this information so as to produce a coherent response at the right time.^2–3 This proposal is supported by numerous experimental results.^4–12

The response of plants to stimuli involves changes in the level of free calcium in the cytosol and such changes exhibit complex spatio-temporal dynamics.^13–20 Using the model of the induction of epidermal meristems in the hypocotyl of flax seedlings,^7–11 our group has shown that when flax seedlings (a few days in age) were subjected to both an abiotic stimulus and a 1 to 3 day-long depletion of calcium in the nutrient medium, they developed numerous meristems over the following fortnight.^7–8 These abiotic stimuli included a manipulation stimulus, wind, drought stress, cold shock, and low intensity electromagnetic radiation over a wide range of frequencies. The results of these experiments are consistent with a central role for calcium in a mechanism responsible for one or more of the processes of registering, storing, recalling, integrating and expressing abiotic information. The nature of the mechanism itself remains unknown. To try to glean an insight into it, we used inhibitors of calcium channels to show that extracellular calcium is implicated and to reveal temporal sensitivities to disruption that depend on the type of abiotic stimulus.

Materials and Methods

Plant growth.

The growth conditions of the plants have already been described.^7–8 Briefly, flax seeds (Linum usitatissimum L., var Ariane) were allowed to germinate for three days, in the dark, on plastic grids fitted on top of growth vessels filled with a modified CERA III nutrient solution;^21–22 then they were allowed to grow, under continuous artificial light (intensity = 32 µmol photons per square meter per second) at a temperature of 23 ± 1°C, using similar plastic grids, growth vessels and growth solution as for germination. The composition of the basic (modified CERA III) nutrient solution and that of other experimental solutions (without calcium, without phosphate, or with interacting pharmacological agents) are given in Table 1. In all cases, the seedlings were set on the plastic grids in such a way that only the roots, or sometimes the roots plus the lower part of the hypocotyls, were in contact with the external solution.

Table 1.

Composition of the various nutrient solutions used in the experiments

Components	Normal Medium	No Ca	No Phosphate	Plus eGTA	Plus RR	Plus La	Plus Gd
Macroelements	KNO3	6.92	11.58	6.92	11.58	6.92	6.92	6.92
(mM)	Ca(NO3)2, 4H2O	2.33	-	2.33	-	2.33	2.33	2.33
	MgSO4, 7H2O	1.62	1.62	1.62	1.62	1.62	1.62	1.62
	NaH2PO4, 2H2O	2.18	2.18	-	2.18	2.18	-	-
	NaNO3	-	-	2.18	2.33	-	2.18	2.18
Microelements	MnSO4, H2O	10	10	10	10	10	10	10
(µM)	CuSO4, 5H2O	1	1	1	1	1	1	1
	ZnSO4, 7H2O	1	1	1	1	1	1	1
	Na2MoO4, 2H2O	0.21	0.21	0.21	0.21	0.21	0.21	0.21
	H3BO3	27.33	27.33	27.33	27.33	27.33	27.33	27.33
	EDTA, Fe3+	100	100	100	100	100	100	100
Pharmacological	EGTA (mM)	-	-	-	2.33	-	-	---
agents	RR (µM)	-	-	-	-	50, 500, 1000	-	-
	La (µM)	-	-	-	-	-	10, 100	-
	Gd (µM)	-	-	-	-	-	-	1, 5, 10

Experimental treatments.

In our present experiments, five types of abiotic signals were studied: a “manipulation stimulus”, drought stress, slow cold treatment, a single cold shock or a double cold shock. In the case of the manipulation stimulus, the germinated seedlings were either (1) sampled and set in the meshes of another plastic grid fitted on top of another growth vessel filled with the same modified CERA III solution as for seed germination (seedlings subjected to the manipulation stimulus) or (2) left to grow in situ, i.e., on the same grid and the same growth vessel where they were germinated (nonstimulated controls). For the drought stress, the seedlings were left for two hours with their roots in open air (out of the nutrient solution). For the slow cold treatment, the seedlings, kept on their plastic grid on top of their growth vessel, were transferred from the normal growth room (23 ± 1°C) to a cold room (4°C, but otherwise identical to the normal growth room); they were left in the cold room for 24 h, and then brought back to the normal room; in the cold room, the fall in temperature (from 23 to 4°C) of the growth medium within the growth vessels took approximately 6 h. For the single cold shock, the seedlings on their plastic grids were transferred for 1 min from their normal growth vessel (23°C) to a vessel with a refrigerated nutrient solution (4°C) and then brought back to their normal growth vessel. For the double cold shock, the plants were subjected to two successive cold shocks with an interval of 1 h between them.

For the calcium depletion, the seedlings on their plastic grids were transferred for 1 to 3 days from their normal growth vessel to a vessel containing a nutrient solution without calcium (4^th column in Table 1), then they were brought back to a vessel with the normal growth solution; in most cases, the calcium deprivation treatment was begun on the second day following the abiotic stimulus. The phosphate deprivation treatment was carried out in a manner identical to that for calcium depletion, except that the seedlings were transferred for up to 1 day from their normal growth vessel to a growth vessel filled with a nutrient solution without phosphate (5^th column in Table 1).

In the experiments with pharmacological agents, the seedlings were transferred for short periods to solutions as indicated in the last four columns of Table 1. EGTA chelates Ca²⁺ with a high specificity relative to Mg²⁺. Ruthenium red (RR), lanthanum (La) and gadolinium (Gd) are primarily, but probably not strictly, inhibitors of calcium channels of the plasmalemma.^14,16–19 The duration of the EGTA-treatment (2.33 µM) was 3 h. The RR-treatments were carried out in the dark, using three different concentrations of the dye (50, 500 and 1000 µM), for a period of 2 or 14 h. The La-treatment (10 or 100 µM) and the Gd-treatment (1, 5 or 10 µM) were given for periods ranging from 1 min to 1 day (La) and 1 h to 1 day (Gd). These pharmacological treatments took place either simultaneously with the abiotic stimulus, or before or after this stimulus with a time interval between stimulus and pharmacological treatment (or between pharmacological treatment and stimulus) in the range of 1 min to 1 h. With the exception of the gadolinium treatment, which tended to cause necrosis of the terminal buds, the treatments by pharmacological agents at the doses used here did not seriously damage the seedlings (not shown).

When the seedlings were transferred from one nutrient medium to another, it was observed using dyes that the combined effect of diffusion and capillarity caused the new solution to pervade the entire hypocotyl almost immediately (not shown). It is thus reasonable to assume that the solutes in the external medium were almost immediately equilibrated with the hypocotyl cell walls. It has been suggested that inhibitors such as La and Gd can be taken up by plant cells and may thus have intracellular effects.¹⁸ Again with dyes, or using the SIMS methodology (for details about this technique, see e.g., Thellier et al. Ref. 23), we have observed (not shown) that ruthenium red and lanthanum penetrated the seedling tissues to some extent, particularly the xylem; but it was not certain that these dyes significantly penetrated into the seedling cells (this is especially the case with La, because the La-concentration which we have used (10 µM) was considerably lower than that used by other authors (10 µM).^14,18–19 EGTA is considered to be membrane impermeable¹⁹ and thus not to penetrate beyond the seedling cell walls.¹⁴

Counting the meristems.

The meristems were counted in the seedlings, when they were at least at the 4-cell stage, within a period ranging approximately from the end of the first week to the end of third week after germination began. Under each experimental condition, on the days chosen for meristem counting, ten seedlings were sampled from each growth vessel and immediately dipped in 50% (v/v) aqueous ethanol. They were left there (24 h or more) until they were sufficiently transparent for the epidermal meristems in the seedling hypocotyls to be counted easily with a conventional light microscope. Each experimental point thus corresponds to the mean value of the numbers of meristems counted in 10 seedlings.

Analysis of the results.

To minimize the effects of the fluctuations on the determination of the mean values of the counted meristems, comparing with one another the curves representing the mean number of meristems per seedling as a function of time was carried out using the curve integral, I, over the total time when the meristems were counted. For each experimental curve, numerical integration was performed by the trapezium method (Fig. 1). The expression of the integral is

$$\text{I=}{\displaystyle \sum _{\text{j=1}}^{\text{f-1}}{\text{(t}}_{\text{j+1}}{\text{-t}}_{\text{j}}{\text{)×(n}}_{\text{j+1}}{\text{+n}}_{\text{j}}\text{)/2}}$$ (1)

in which t₁ and t_f are the initial and final times (days) when meristems were counted, t_j and t_j+1 are two successive times when meristems were counted, and n_j and n_j+1 are the corresponding mean numbers of meristems counted per seedling. Apart from minimizing (by integration) the role of the fluctuations in the mean values of the number of meristems, this procedure has the advantage of characterising meristem production with a single parameter, I.

Figure 1.

An example of a curve representing the mean number of meristems per seedling, n, as a function of the time, t (days), when the meristems were counted, and calculation of the integral, I, of this curve. In this case, a manipulation stimulus (vertical arrow) was applied at the end of the third day after germination began and calcium depletion (- Ca) occurred immediately after the manipulation stimulus and lasted for 1 day. The mean number of meristems per seedling increased from 0 (6^th day) to a little more than 13 (18^th day). For the calculation of the integral, I, t₁, t₂, …, t_j, t_j+1, …, t_f (index f = final) are the times (days) when the meristems were counted, n₁, n₂, …, n_j, n_j+1, …, n_f are the corresponding values of the mean numbers of meristems counted per seedling, and A₁, A₂, …, A_j, A_j+1, …, A_f are the corresponding experimental points on the curve. The surface area of the quadrilateral t_jt_j+1A_j+1A_j is equal to (t_j+1 − t_j)·(n_j+1 + n_j)/2 and the integral, I (see equation 1), of the curve is obtained by summing up the surface areas of all the quadrilaterals from t₁t₂A₂A₁ to t_f−1t_fA_fA_f−1.

Results

The results appear in Figures 1–10. The code for Figures 2–10 is as follows. Each experimental condition is numbered {1}, {2}, {3}, etc. Time zero corresponds to the beginning of germination of the flax seeds; the age of the seedlings when the various treatments were carried out is indicated (1, 2, 3, etc.), in days, at the top of each figure. The symbols for the abiotic stimuli and the pharmacological agents are indicated in Table 2. When treatments by pharmacological agents took place, the concentration of each agent and the duration of the treatment are indicated in the figures or in the figure captions. When abiotic stimulus and pharmacological treatment were not given simultaneously, the time between them (e.g., (1 min), (2 min), etc.) is shown immediately below. The I-value, together with its value relative to that of the positive control (abiotic stimulus followed by calcium depletion) is also given (%) for each experimental condition.

Figure 10.

Effect of EGTA, ruthenium red (RR) and lanthanum (La) on flax seedlings subjected to cold shock or to double cold shock. Three successive experiments have been carried out (lines {1} to {3}, {4} to {14} and {15} and {16}, respectively). The abiotic stimuli (cold shock in most cases) and the pharmacological treatments were applied on the beginning of the 5^th day and calcium depletion took place on the 7^th and 8^th days. Line {2} corresponds to a nonstimulated, calcium-depleted control and lines {1}, {3}, {4} and {15} to stimulated and calcium-depleted controls (stimulation = manipulation stimulus in line {1} and cold shock or double cold shock in lines {3}, {4} and {15}). EGTA treatment was 2.3 µM during 3 h; cold shock was applied either at the end (line {5}) or at the beginning (line {6}) of the EGTA-treatment and there was sometimes a delay of 1, 2 and 5 min between cold shock and EGTA treatment (lines {7} to {9}, respectively). The ruthenium red (RR) treatment (lines {10} and {16})was 0.5 mM during 2 h; a cold shock (line {10}) or a double cold shock (line {16}) was given at the end of the RR-treatment. The La-treatment was 10 µM during 4 h; a cold shock was applied at the beginning of the La-treatment (line {11}) or 1, 2 or 5 min before the La-treatment (lines {12} to {14}, respectively.) The meristems were counted in the time interval of days 9–29.

Figure 2.

Effect of EGTA on flax seedlings subjected to manipulation stimulus. EGTA was applied at the dose of 2.33 µM during 3 h (lines {3} to {7}). The EGTA-treatment was begun at either the moment when the manipulation stimulus was carried out (line {3}) or after an interval of time of 2 min to 1 h (lines {4} to {7}). Controls appear in lines {1} and {2}. The meristems were counted in the time interval of days 6–21.

Table 2.

Meaning of the symbols and abbreviations used in the figures

Symbols and Abbreviations	Meaning
	Manipulation stimulus
SCT	Slow cold treatment
	Cold shock
	Double cold shock
	Drought stress
	Time when calcium depletion occurred
EGTA	Ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid
RR	Ruthenium red
La	Lanthanum
Gd	Gadolinium

Effects of the depletion of different ionic species and of different abiotic stimuli.

A significant increase in the number of meristems resulted from subjecting the flax seedlings to a manipulation stimulus followed by a temporary depletion of calcium. A typical example is shown in Figure 1 in which the integral, I, of the curve was equal to 111. Numerous experiments (data not shown) showed that, for plants subjected to a manipulation stimulus followed by calcium depletion, the I-value was usually in the range of 50 to 100 and sometimes much more, while the I-value for seedlings subjected to only the manipulation stimulus or to only the calcium depletion was usually in the range of 10 to 25 or even less. There is also a seasonal effect⁸ and the I-values are particularly high, both for the stimulated seedlings and for the nonstimulated controls, when the experiments are carried out at the end of spring or beginning of summer.

The nutrient media containing La and Gd did not contain phosphate since La or Gd phosphate precipitates at the concentrations used in our experiments. We therefore ensured that depleting the medium in phosphate did not induce the formation of meristems after a stimulus in the absence of La or Gd (data not shown). Depleting magnesium, sodium or potassium from the media (in association with an abiotic stimulus) did not result in meristem production, as previously reported;⁸ indeed, we have never found any treatment other than calcium depletion that could do this in the hypocotyls of noninjured and nonhormone treated flax seedlings.

The effect of the slow cold treatment (SCT) was less than that of the manipulation stimulus (compare for instance the I-values in lines {1} and {4} in Fig. 4) whilst the effect of cold shock was similar to that of the manipulation stimulus (compare for instance the I-values in lines {4} and {1} of Fig. 10); it should be noted that the effect of a drought stress was also similar to that of a manipulation stimulus.⁸ The effect of a double cold shock appeared to be slightly greater than that of a single cold shock (compare for instance the I-values in lines {15} and {4} again in Fig. 10). This is not surprising since giving several stimuli usually has a cumulative effect.^8,14

Figure 4.

Effect of ruthenium red (RR) on flax seedlings subjected to a manipulation stimulus or a slow cold treatment (SCT). Two successive experiments were carried out, corresponding to lines {1} to {3} (first experiment) and lines {4} to {6} (second experiment). Calcium depletion (3 days) occurred 3 days after the manipulation stimulus or 2 days after the SCT. RR was supplied at concentrations of 50 µM (lines {3} and {5}) or 1 µM (line {6}) during 2 h, immediately before the abiotic stimulus, whether this stimulus was due to manipulation (line {3}) or was a SCT (lines {5} and {6}). Since the seedlings were kept in the dark during the RR-treatment, the non-RR treated controls (lines {1}, {2} and {4}) were kept in the dark accordingly. The meristems were counted in the time interval of days 11–28.

From a number of preliminary experiments (data not shown) it appeared that the duration of the temporary depletion of calcium in the range of 1–3 days had no significant effect on the meristem response. The length of time between an abiotic stimulus and calcium depletion had no significant effect on the I-value in the meristem response (for example, in (Fig. 10) compare the I-values in line {3} and line {4}).

Effect of EGTA on flax seedlings subjected to a manipulation stimulus.

In the absence of EGTA (line {1}), seedlings subjected to manipulation stimulus plus calcium depletion had an I-value of 49 (Fig. 2). The addition of EGTA into the nutrient solution during calcium depletion (line {2}) did not appreciably change this result. Subjecting the seedlings to EGTA-treatment severely inhibited the response to the manipulation stimulus (I-value equal to 12, i.e., 24% of the value of the non-EGTA treated control) when this stimulus was applied to the seedlings in the presence of EGTA (compare lines {1} and {3}), whereas EGTA had no effect (I-values not significantly different from 50, or even slightly larger) when applied shortly (in the range of 2 min to 1 h) after the manipulation stimulus (compare lines {4} to {7} with line {1}). This would be consistent with the availability of external calcium for 2 min after the stimulus being sufficient for the information processing leading to the meristem response. By contrast, the addition of EGTA did not change the reaction of tendrils to touch in Bryonia plants.¹⁷

Effect of ruthenium red on flax seedlings subjected to manipulation stimulus, slow cold treatment or drought stress.

A set of experiments (Fig. 3) showed that the response to the manipulation stimulus was completely inhibited by an RR-treatment (50 µM for 14 h) providing the stimulus was given at the end of the RR-treatment (compare the I-values in lines {1} and {2}). However, when the RR-treatment was given at the end of calcium depletion, its inhibitory effect was much reduced although not absent since the I-value (line {4}) was close to 60% of that of the non-RR treated control (line {3}).

Figure 3.

Effect of ruthenium red (RR) on flax seedlings subjected to manipulation stimulus. Two successive experiments were carried out, corresponding to lines {1} and {2} (first experiment) and lines {3} and {4} (second experiment). Calcium depletion (3 days in this case) occurred immediately after the manipulation stimulus. Ruthenium red was supplied (lines {2} and {4}) at the concentration of 50 µM during 14 h, either immediately before the manipulation stimulus or immediately after calcium depletion. Since the seedlings were kept in the dark during the RR-treatment, the non-RR treated controls (lines {1} and {3}) were kept in the dark accordingly. The meristems were counted in the time interval of days 6–26.

An RR-treatment (RR at 50 µM) for only 2 h given immediately before a manipulation stimulus was enough to totally inhibit the response (in Fig. 4, compare the I-value in line {3} with those in line {1} (positive control) and line {2} (negative control)). The effect of a 2 h RR-treatment on the response to the SCT was qualitatively similar to that to the manipulation stimulus, except that an RR concentration of 1 µM was needed to totally inhibit the response to the SCT (compare the I-value in line {6} with that in line {4}) and a concentration of 50 µM caused only partial inhibition (compare the I-value in line {5} with that in line {4}).

Another set of experiments (Fig. 5) was performed at a different time of the year, mid-May (which is the likely explanation for the unusually high I-values in this figure, see § Effect of depletion of different ionic species and of different abiotic stimuli). The various controls (in the absence or in the presence of an RR-treatment) shown in lines {1} to {6} confirm that only the association of an abiotic stimulus (here a manipulation stimulus) with a temporary depletion of calcium (line {1}) caused a maximal production of meristems since meristem production was considerably less in the absence of an abiotic stimulus (lines {2}, {3}, {5} and {6}) or in the absence of a temporary calcium depletion (lines {3} and {4}). In the cases when an abiotic stimulus was given before a temporary calcium depletion, an RR-treatment (50 µM during 14 h) inhibited meristem production, whether the abiotic stimulus was a manipulation stimulus (lines {7} to {9}) or a drought stress (lines {11}) and whether this abiotic stimulus was applied at the end (lines {7} and {11}) or at the beginning (line {8}) of the RR-treatment or else 1 day before beginning the RR-treatment (line {9}). It is known⁸ that the combined effect of calcium depletion with an abiotic stimulus is the same whether calcium depletion occurs after or before the abiotic stimulus. However, when the RR-treatment was carried out after temporary calcium depletion and before a manipulation stimulus (line {10}), it is not possible to decide whether the lack of meristem production was due to the inhibition by RR of the effect of the manipulation stimulus or to that of the temporary calcium depletion.

Figure 5.

Effect of ruthenium red (RR) on flax seedlings subjected to a manipulation stimulus or a drought stress. Calcium depletion was applied for 2 days. RR was supplied at a concentration of 50 µM during 14 h either immediately before or immediately after the abiotic stimulus. Since the seedlings were kept in the dark during the RR-treatment and this RR-treatment took place at the end of the 5^th day in most cases, the non-RR treated controls (lines {1} and {2}) were kept in the dark accordingly. The meristems were counted in the time interval of days 7–26.

The essential result of this series of experiments is that RR completely inhibits the meristem response to a manipulation stimulus or a drought stress, even at a low concentration (50 µM), but that a much higher concentration (1 µM) is needed to completely inhibit the response to an SCT.

Effect of lanthanum on flax seedlings subjected to manipulation stimulus.

A La-treatment (10 µM) inhibited the meristem response to a manipulation stimulus and longer La-treatments were more inhibitory (compare lines {2} to {4} to line {1} and line {6} to line {5} in Fig. 6). This La-treatment became totally ineffective when it was delayed by 2 to 60 min relative to the manipulation stimulus (compare the I-values in lines {9} to {12} to those in lines {7} and {8} in Fig. 6).

Figure 6.

Effect of lanthanum (La) on flax seedlings subjected to a manipulation stimulus. Three different experiments were carried out. In all cases, a manipulation stimulus was performed at the end of the 3^rd day and calcium depletion (2 days) occurred during days 6 and 7. The non-La treated controls appear in lines {1}, {5} and {7}. In the first experiment (lines {1} to {4}), a La-treatment (10 µM) was applied immediately after a manipulation stimulus for 10 min, 30 min and 3 h (lines {2} to {4}). In the second experiment (lines {5} and {6}), a La-treatment (10 µM) was applied immediately after a manipulation stimulus for 3 h (line {6}). In the third experiment (lines {7} to {12}), a La-treatment (10 µM) was applied for 3 h either immediately after a manipulation stimulus (line {8}) or after increasing intervals of time (2, 5, 10 and 60 min) after the manipulation stimulus (lines {9} to {12}). The meristems were counted in the time interval of days 6–21 in the three experiments.

For a part, the data in Figure 7 are consistent with those in Figure 6 since a long La-treatment (10 µM for 1 day) begun at the moment when a manipulation stimulus was given strongly inhibited the meristem response (compare the I-value in line {3} to those in lines {1} (positive control) and {2} (negative control) and the I-value in line {6} with that in line {5} in Fig. 7). However, in stark contrast with the data in lines {7} to {12} in Figure 6, when the La-treatment was begun one day after the manipulation stimulus (and immediately before the temporary calcium depletion), the meristem production was only reduced to 68% of that of the non-La treated control (compare line {4} with line {1} in Fig. 7). The explanation for this apparent discrepancy between the data in the two figures might be that a long-lasting (1 day) La-treatment either progressively erased the meristem-production instruction memorized within the seedlings as a consequence of the manipulation stimulus or tended to abolish the effect of calcium depletion.

Figure 7.

Complementary data on the effect of lanthanum (La) on flax seedlings subjected to a manipulation stimulus. Two different experiments have been carried out (lines {1} to {4} and lines {5} and {6} for the first and second experiment, respectively). Calcium depletion (2 days) took place either during days 5–6 or 6–7. Control seedlings were subjected to manipulation stimulus followed by temporary calcium depletion (lines {1} and {5}). The La-treatment (10 µM during 1 day) occurred on nonstimulated seedlings (line {2}), or immediately after the manipulation stimulus and before the temporary depletion of calcium (lines {3} and {6}), or else 1 day after a manipulation stimulus and immediately before calcium depletion (line {4}). The meristems were counted in the time interval of days 7–25 in both experiments.

Effect of gadolinium on flax seedlings subjected to manipulation stimulus.

A Gd-treatment (1 µM for 4 h) effectively inhibited the meristem response to a manipulation stimulus irrespective of whether the Gd-treatment was given after (line {7} in Fig. 8) or before (lines {4} and {10}) the stimulus. All the Gd-treatments inhibited the meristem response significantly, and the more concentrated and the longer the Gd-treatment, the more intense was the inhibition (lines {4}, {6} to {10} and {12} to {14}). Controls subjected to a manipulation stimulus followed by a temporary calcium depletion (shown in Fig. 8 as lines {1} (first experiment), {5} (second experiment) and {11} (third experiment)) gave results consistent with those reported above, as did a nonstimulated noncalcium depleted control (line {2}) and a lanthanum-treated control (line {3}).

Figure 8.

Effect of gadolinium (Gd) on flax seedlings subjected to manipulation stimulus. Three successive experiments have been carried out (lines {1} to {4}, {5} to {10} and {11} to {14}, respectively). Calcium depletion (2 days) took place during days 6–7. Control seedlings were subjected to manipulation stimulus followed by calcium depletion (lines {1}, {5} and {11}) or to neither a manipulation stimulus nor the calcium depletion (line {2}). A single La-treatment was carried out for comparison with the data in Figures 6 and 7. For each type of gadolinium treatment (lines {4}, {6} to {10} and {12} to {14}), the Gd-concentration and the treatment-duration are indicated in the figure. The meristems were counted in the time interval of days 6–22.

Effect of lanthanum on flax seedlings subjected to cold shock.

A 10 µM La-treatment effectively inhibited the meristem response to a cold shock provided that the La-treatment lasted at least 10 min [(in Fig. 9, compare the I-values in lines {3} to {6} to those of the non-La treated control (line {1}) and of the non-La treated, nonstimulated control (line {2})]. The inhibitory effect characteristic of a La-treatment of a given length was little changed by giving the treatment before or after the cold shock (compare the I-values of lines {7} to {10} to those of lines {3} to {6}, respectively). A delay of 1 min between cold shock and La-treatment (line {11}) or between La-treatment and cold shock (line {12}) did not change the effect of the La-treatment significantly.

Figure 9.

Effect of lanthanum (La) on flax seedlings subjected to cold shock. In this experiment, flax seedlings were subjected on the beginning of the 6^th day to 10 µM La-treatments of various durations (1 to 30 min), to a cold shock occurring at the beginning (lines {3} to {6}) or at the end (lines {7} to {10}) of the La-treatment and with a possible delay of 1 min between cold shock and La-treatment (line {11}), or between La-treatment and cold shock (line {12}) and to temporary calcium depletion on the 7^th and 8^th days. The non-La treated controls subjected or not to cold shock appear in lines {1} and {2}, respectively. The meristems were counted in the time interval of days 9–29.

Effect of EGTA, ruthenium red and lanthanum on flax seedlings subjected to cold shock or to double cold shock.

EGTA (2.33 µM, 3 h) caused a significant inhibition of the meristem response to cold shock (in Fig. 10, compare the I-values in lines {5} and {6} with that in line {4}); that said, this inhibition was incomplete (compare the I-values in lines {5} and {6} with that in line {2}). Moreover, delaying the EGTA-treatment by 1, 2 and 5 min relative to the cold shock still caused a partial inhibition of this response (compare the I-values in lines {7} to {9} with that in line {4}).

An RR-treatment (0.5 µM for 2 h) totally inhibited the response to a single cold shock [in Fig. 10, compare the I-value in line {10} with those of the stimulated, calcium-depleted control (line {4}) and of the nonstimulated, calcium-depleted control (line {2})]. The same RR-treatment inhibited the response to a double cold shock almost as effectively as it did to a single cold shock [compare the I-value in line {16} with those of the stimulated, calcium-depleted control (line {15}) and of the nonstimulated, calcium-depleted control (line {2})].

A La-treatment (10 mM for 4 h) strongly inhibited the meristem response to a cold shock [in Fig. 10 compare the I-value in line {11} with those of the stimulated, calcium-depleted control (line {4}) and of the nonstimulated, calcium-depleted control (line {2})]. Delaying the La-treatment by 1, 2 and 5 min relative to the cold shock still caused a partial inhibition of the meristem response (compare the I-values in lines {12} to {14} with that in line {4}).

Discussion

Flax seedlings produce an increased number of meristems in response to a variety of abiotic stimuli provided these are followed by a calcium deprivation step. This meristem response constitutes a powerful system for studying the processing of abiotic information by plants. Among the various stimuli to which flax seedlings were exposed in the present investigation, only the manipulation stimulus is a mechanical one.

When a manipulation stimulus was followed immediately by an EGTA treatment, the meristem response to the manipulation was severely inhibited (I-value = 24% of the non-EGTA treated control as seen by comparing lines {1} and {3} in Fig 2). RR, La and Gd also severely inhibited the response to a manipulation stimulus (Figs. 3–8). Despite doubts about these agents penetrating cells and about the general toxicity of Gd (see § Experimental treatments), these results are largely consistent with the idea that calcium entry (which would be inhibited by the agents) is associated with the processing of the manipulation stimulus. The idea that calcium entry is an important step is also supported by the fact that RR Figure 5 or Gd Figure 8) addition immediately after a manipulation stimulus (RR or Gd post-treatment) was almost as efficient as RR or Gd pretreatment to inhibit the meristem response [compare e.g., line {8} with line {7} in (Fig. 5) and line {7} with line {10} in Fig. 8)], when these agents did not have time to penetrate the seedling cells and presumably affected only calcium channels in the plasma membrane at the moment when the stimulus was given in the case of the post-treatment. When an EGTA treatment (Fig. 2) or a La treatment (Fig. 6) was delayed by 2 min or more, relative to a manipulation stimulus, these pharmacological agents no longer inhibited the response to this stimulus. This is consistent with the idea that there is a short period (2 min at the most) during which the storage of the information leading to meristem production is vulnerable but that after this period the information becomes insensitive to these pharmacological agents.

In the case of the nonmechanical, abiotic stimuli (drought stress (Fig. 5), slow cold treatment (Fig. 4), single cold shock (Figs. 9 and 10) and double cold shock (Fig. 10), subsequent addition of EGTA, RR and La again inhibited meristem production depending on the concentration and length of exposure to these agents. This is therefore consistent with entry of external calcium also being involved in the processing of nonmechanical stimuli. Indeed, experiments on whole plants transformed with aequorin have shown that both mechanical stimulation and cold shock resulted in a transient increase in cytosolic calcium due to entry of external calcium.^13,16–17 However, when a La treatment (Figs. 9 and 10) was delayed by up to 5 min relative to a cold shock, the effect of the pharmacological agent was only partially suppressed (I-value of the order of 50% instead of 100%). The initial stages of information processing following cold shock are thus rapid but nevertheless significantly slower than those following a mechanical stimulus such as manipulation.

The mechanism(s) responsible for the registration, storage, integration and recall aspects of the long-term processing of abiotic stimuli are still unclear. Interestingly it has been found²⁰ that the duration of the pulse of cytosolic calcium is significantly shorter for a mechanical stimulus (0.3 min) than for a cold shock (1.5 min). This may be relevant to our finding that the period of vulnerability of information processing is longer for cold shock than for mechanical (manipulation) stimuli. Previously, we have invoked the possibility that the condensation of calcium^24–26 on charged filaments in the cytoplasm and nucleus at a critical value of the charge density might play a key role in these mechanisms.²⁷ This raises the question of whether the rapid step of information processing occurring after mechanical stimulation involves, for example, fast-acting protein kinases and/or phosphatases on the cytoskeleton whilst the longer period needed for processing other abiotic stimuli involves, for example, a slower effect of similar enzymes to modify the charge density of chromatin. In both cases, condensation of ions such as calcium might constitute part of a unifying mechanism for long-term information processing.

Conclusion

Calcium appears to be necessary for the processing of information about a variety of abiotic stimuli by flax seedlings, providing the seedlings are subjected to a temporary depletion of calcium. It is likely that this processing, which leads to meristem production, depends on a transient entry of calcium into the cytoplasm of the seedlings as with other plant systems. The period of vulnerability of the meristem response is much shorter following a manipulation stimulus (and possibly other mechanical stimuli) than following cold shock (and possibly other nonmechanical stimuli).

Abbreviations

EDTA

ethylenediaminetetraacetic acid

EGTA

ethylene glycol tetraacetic acid

RR

ruthenium red

SCT

slow cold treatment