Movements of Cassia fasciculata - NCBI

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effect induced by chelatants (EDTA and EGTA) (4, 13, 19) and the promotive ... 17) wrapped in green plexiglass (Micap 3074, France) at a fluence rate of 15 ...
Plant Physiol. (1989) 90, 697-701

Received for publication August 10, 1988 and in revised form January 15, 1989

0032-0889/89/90/0697/05/$01 .00/0

Effects of Compounds Affecting Calcium Channels on Phytochrome- and Blue Pigment-Mediated Pulvinar Movements of Cassia fasciculata' Gabriel Roblin*, Pierrette Fleurat-Lessard, and Janine Bonmort University of Poitiers, Station Biologique de Beau-Site, 25 Faubourg Saint-Cyprien, F-86000, Poitiers, France ABSTRACT

MATERIALS AND METHODS Plant Growth Conditions

Verapamil and nifedipine, known as calcium channel blockers, inhibited the phytochrome-mediated movements induced on Cassia fasciculata leaflets by a light-off signal, whereas they had no effect on the 'blue' pigment-mediated movements induced by a light-on signal. LaCI3 inhibited both types of reactions, but the inhibition of light-induced opening needed a 10 times higher concentration than that of dark-induced closure. Bay K 8644, an activator of calcium channels, increased the rate of dark-induced closure, whereas it had no effect on the light-induced opening. These data suggest that calcium ions are not mobilized in the same way in the two types of movements: possibly from extemal stores in the phytochrome-mediated reacffon and from intemal stores in the 'blue' pigment-mediated reaction.

Plants of Cassiafasciculata Michx. were grown from seeds in a mixture of garden-earth, heath-mold, sand and peat (3:2:3:2 by volume). They were maintained in climate-controlled chambers at 26 ± 1°C and 65 ± 2% RH. White fluorescent light (Phytor LF 40 ACEC Charleroi, Belgium) was given at a photon fluence rate (400-700 nm) of 36 Mmole m-2s- at the apex of the plants according to two daily schedules. In set A, the light was provided from 7 to 21 h (normal photoperiodic cycle), and in set B, light was provided from 19 to 9 h (inverted photoperiodic cycle). Experimental Light Sources

The source for the blue light was high pressure mercury lamps (Philips SP 500 W); the emitted light was filtered through a blue glass filter (BG 37, 4 mm, Schott and Gen, Mainz, FRG) giving a fluence rate of 30 ,mol m-2s-'. Green light was provided by green fluorescent tubes (Philips TF 40/ 17) wrapped in green plexiglass (Micap 3074, France) at a fluence rate of 15 gmol m-2s-'. Yellow light was obtained from a halide reflector bulb (Osram 15 V, 150 W), light from which was filtered through a yellow glass (OG 515, 4mm, Schott and Gen, Mainz, FRG) giving a fluence rate of 96 Mmol m-2s-'. R2 light was given by red fluorescent tubes (Philips TF 40/15) at a fluence rate of 15 jsmol m-2s-I and FR light was given by incandescent lamps (Claude 500 W) filtered through a FR glass (RG N9 Schott and Gen, Mainz, FRG) giving a total fluence rate of 150 ,umol m-2S-1 (30 ,umol m-2s-' being noted in the range 700-800 nm). The fluence rate was measured at the leaf level using a thermopile (Hilger-Schwartz FT 16-1).

In various plants, particularly in Leguminosae, leaf movements can be induced by transferring leaves from light to darkness or from darkness to light. The kinetics, energetics, and some reaction steps of these movements are beginning to

be well known (22, 23). These movements, brought about by motor organs (pulvini), are not the result of growth processes but involve reversible turgor variations driven by ionic migrations, namely K+ and Cl-, in the cortical parenchyma cells of the pulvini (24). Furthermore, on the basis of the inhibitory effect induced by chelatants (EDTA and EGTA) (4, 13, 19) and the promotive effect induced by the ionophore A 23187 (19), calcium is hypothesized to intervene in the regulation of the pulvinar movements. Results presented previously (19, 20) suggest that calmodulin could be implicated in some way. The question raised now is to determine in what way the calcium ions are mobilized before forming the active complex with calmodulin and, in particular, from what stores they could be liberated following a stimulus. This problem is considered in the present investigation by use of compounds known to affect calcium channels in animal cells either through an inhibition (LaCl3, verapamil, nifedipine) or a promotion (Bay K 8644). Indeed, the existence of Ca2" channels in plants has been suggested by the identification of binding sites for Ca2" channel blockers (1, 5, 10).

Experimental Procedures Leaves of C. fasciculata Michx. were excised at 9 h from plants 4 months old and possessing about 20 well-developed leaves. The cut petioles of the leaves were dipped in distilled H20 for 2 h to allow recovery from shock. After this period, the leaves were transferred to test solutions for 3 h or maintained in distilled water in the controls. Then, they were put either in the dark in the middle of the photophase (14 h) for the leaves in normal photoperiodic cycle or in the light in the

'Supported by the Centre National de la Recherche Scientifique (URA 81).

2 Abbreviations: R, red; FR, far red. 697

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ROBLIN ET AL.

middle of the nyctophase (14 h) for the leaves in inverted photoperiodic cycle. This schedule was chosen to avoid interferences with the well-known endogenous rhythmic behavior of the pulvini evidenced by leaflet closure in plants maintained in light at the end of the photophase (set A) and by leaflet opening in plants maintained in darkness at the end of the nyctophase (set B). These movements linked to a rhythmic component were observed in our general experimental conditions at about 17 h in each case (set A and set B), at a time when the darkand light-induced movements were achieved. Expression of Results The pulvinar movements were followed by measuring the distance between the leaflet tips with a caliper square. This linear measurement was then converted into angular values as previously described (3). Curves represent the mean values obtained from the sum of separate experiments on eight individual leaves.

Chemicals Verapamil and nifedipine were purchased from Sigma and LaCl3 from Merck. LaCl3 and verapamil (hydrochloride) were dissolved in deionized water, whereas nifedipine and Bay K 8644 were dissolved in DMSO. In these experiments, the final DMSO concentration was fixed at 1 % at the highest concentration of the product tested. Controls received the same final DMSO concentration which has been observed to be ineffective on the leaflet movements (18). The pH of the solutions was brought to 5.0 with 0.1 N KOH or 0.1 N HCI. In all the experiments, a necrosis was never observed following application of the compounds in the cited concentration range.

RESULTS Verification That Phytochrome and a Blue Absorbing Pigment Are Respectively Involved in the Dark- and Light-induced Leaflet Movement in C. fasciculata Figure IA shows that leaflets of C. fasciculata irradiated with a R source for 5 min before the light-off signal closed in darkness. By contrast, irradiation with a FR source for 5 min hindered the dark reaction. The responses to an alternation of R and FR irradiations showed that the leaflets remained open if exposure to FR was last, whereas they closed normally if R exposure was last. It can be noted that closure in controls (leaflets darkened without preirradiation) occurred at the same rate as in the R preirradiated leaflets, indicating that the phytochrome is found in controls in a photostationary state allowing a complete reaction as in the R preirradiated pulvini. Figure lB presents the sensitivity under various light sources for opening response of leaflets irradiated in the middle of the nyctophase of the photoperiodic cycle. In these conditions, the blue part of the spectrum contains the active radiations. A small reactivity was also noted under the FR source, however much lower (9%) than the blue response. Thus, when the leaflets were stimulated by white light in further experiments, the reaction was ascribed to the blue part of the spectrum.

Plant Physiol. Vol. 90,1989

Effects of Antagonists of Calcium Channels, LaCI3, Verapamil, and Nifedipine

As can be seen in Figure 2A, LaCl3 disturbed both the darkand light-induced movements. It can be emphasized that the dark-induced closure was inhibited at concentrations higher than 1 x l0-' M, whereas the light-induced opening was only inhibited at concentrations higher than 1 x l0-3 M. Treatment of the leaves with verapamil and nifedipine resulted in inhibition of the dark-induced closure; by contrast, the same treatment produced no marked effect on the lightinduced opening (Fig. 2, B and C). An interesting point is that verapamil showed this inhibitory effect at concentrations higher than 1 x l0-5 M, whereas nifedipine was effective at concentrations higher than 1 x 10-6 M. It is also noteworthy that nifedipine induced a weak promotion of the light-induced movement at the highest concentration tested (e.g. 1 x 10 M). The data in Figure 3 show that these compounds inhibited the dark-induced closure without destroying the phytochrome response; it can be noted that inhibition of closure by these calcium antagonists and FR irradiation was additive. Effects of an Agonist, Bay K 8644, of Calcium Channels As can be seen in Figure 4, Bay K 8644 induced a dual effect on the dark-induced movement according to the applied dose: at higher concentration (1 x 10-4 M), this product induced a strong inhibitory effect, whereas at lower concentrations it increased the rate of leaflet closure, the maximal effect being noted at 1 x 10-6 M. Additional experiments (Fig. 3) show that phytochrome modulated this response, since irradiations by R and FR before darkness altered the leaflet closure activated by this compound. It was also observed that the light-induced opening was slightly promoted at 1 x l0-4 M, the product having no marked effect at the other concentrations tested (Fig. 4).

DISCUSSION

Leaflet movements of various plants can be induced by transferring leaves from light to darkness or from darkness to light. These movements are the result of ionic migrations (namely K+ and Cl-) occurring concomitantly with water fluxes into or out of motor cells (24). Previous observations from other plants such as Mimosa pudica (7) and Albizzia julibrissin (11, 13, 23) and the results reported in Figure IA from C. fasciculata show that changes in the photostationary state of phytochrome are an important step mediating pulvinar movements when leaves are transfered from light to darkness. The data presented in Figure 1B, in agreement with previous results (8, 1 1), show that a blue and FR absorbing pigment (cryptochrome?) is the photoreceptor triggering the light-induced pulvinar movements. It can be noted that the dark-induced closure occurs only some minutes after the lightoff signal, whereas the light-induced opening needs a lag-time of 30 to 45 min even at high light irradiation. The facts that the photoreceptor involved in each case and that the kinetics of the induced movements are not the same (see Fig. 1 for example) suggest that the underlying mechanism is not purely reverse.

CALCIUM AND LEAF MOVEMENTS

A

B

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RI D -I -I

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B W

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w 50 -L

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Figure 1. Leaflets movements in C. fasciculata (A) induced on open pulvini by darkness following 5 min R, FR irradiation or alternation of both and (B) induced on closed pulvini by continuous irradiation under different light sources, white (0 W), blue (* B), green (A G), Yellow (A Y), R (0) and FR (U). Vertical bars, SE; N, 16. D, the light is switched off in the middle of the photophase of the photoperiodic cycle; L, the light is switched on in the middle of the nyctophase of the photoperiodic

cycle.

Y

-i