The effect of external Ca²⁺ and Ca²⁺-channel modulators on red-light-induced swelling of protoplasts of Phaseolus radiatus L.

Abstract

Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean (Phaseolus radiatus L.) was observed only when Ca²⁺ ions were present in the medium. The optimal CaCl₂ concentration was 250μM. Swelling response declined when Ca²⁺ was supplied into the medium after red light irradiation. The Ca²⁺chelator EGTA eliminated the red-light-induced swelling and ⁴⁵Ca²⁺ accumulation in the protoplasts. In contrast, A₂₃₁₈₇, a Ca²⁺-ionophore, could mimic the effect of red light in darkness. These results indicate that Ca²⁺ may play a role in light signal transduction. In addition, swelling response was prevented by TFP and CPZ (both are CaM antagonists), implying the involvement of CaM in red-light-induced and Ca²⁺ -dependent protoplast swelling.

Introduction

Most of the processes in plant growth and development depend on the presence of calcium ions. During the past few decades, it has been demonstrated that calcium, as a ‘secondary messenger’, participates the transduction of environmental signals^{1, 2, 3, 4, 5}. The hypothesis that a phytochrome-induced increase in intracellular Ca²⁺ concentration is necessary for the promotion of some phytochrome responses was supported by two best characterized phytochrome responses: light chloroplast rotation in the alga, Mougetia⁶ and spore germination in the fern, Onoclea⁵. In monoco plants such as Avena sativa, Triticum aestivum, Zea may and Vallisneria, etiolated leaf protoplasts swelled in response to red light^{7, 8, 9}. Red light increased the rate of ⁴⁵Ca²⁺ uptake by corn leaf protoplasts, and the response was reversed by far-red light⁷. Recent evidence has shown that this physiological response was preceded by a transient increase in cytosolic free calcium¹⁰ and illumination of the protoplast increased the open probability of ion channel¹¹. Little evidence has been obtained so far in dico plants, except that chloroplasts and protoplasts from spinach leaves exhibited a light-induced Ca²⁺ influx using the metallochromic indicator arsenazo III^{12, 13}. In our previous reports^{14, 15}, we have shown that phytochrome induced swelling of protoplasts of mung bean hypocotyls. The study reported here was designed to elucidate the role of calcium ions in the red-light-induced swelling of protoplasts of Phaseolus radiatus L.

Materials and Methods

Plant material

Seeds of mung bean (Phaseolus radiatus L.) were surface sterilized with 70% ethanol for 1 min and 0.1% HgCl₂ for 5 min, then washed with sterile water (3 changes). Sterilized seeds were grown in darkness at 25± 1°C. Etiolated seedlings were harvested after 64 h.

Protoplast isolation

Hypocotyl segments (5 mm in length) were excised from 3 mm below the hook, then cut transversely into 0.5 mm slices, and plasmolysed (1 h) in a solution of 13% (w/v) mannitol containing KH₂PO₄ 27.2, KNO₃ 101, CaCl₂·2H₂O 1480, MgSO₄·7H₂O 240, KI 0.16, CuSO4·5H₂O 0.025 mg/L, (pH 5.6). Plasmolysed tissue from 50 hypocotyls were incubated on a rotary shaker (50 ∼ 60 r/min, 25 ± 1 °C) for 5 ∼ 6 h in an enzyme solution containing 2% (w/v) cellulase, 1% (w/v) hemicellulase, 0.5% pectinase, 5 mM MES, 9% (w/v) mannitol and the other salt components as used for plasmolysis (pH 5.2). The enzyme-protoplasts mixture was filtered through a nylon sieve (64 μM pore size), and centrifuged (100 × g, 10 min). The pellet was suspended in a 21% (w/v) sucrose solution (other salt components were same as plasmolysis solution) and centrifuged (100 × g, 5min). Floating protoplasts were washed twice with a solution of 9% mannitol containing MES 5 mM , with or without CaCl₂ 250 μM, pH 5.6. The final pellet was suspended to the desired concentration (10⁵ to 5 × 10⁵ cells per ml) in a suspension medium containing 9% (w/v) mannitol, MES 5 mM. Protoplast quantification was performed using haemocytemeter and all protoplast suspension used showed > 95% viability as assessed by fluorescein diacetate (FDA) staining¹⁶. All manipulations were performed under a dim green light (<0.2 Wm⁻²).

In all experiments, performed in the present work, protoplasts were maintained in darkness or irradiated with 3 min red light (10.5 Wm⁻²), then incubated for 30 min in darkness at 20±2°C before volume measurement.

Light sources

The red light source was obtained as described by Long et al¹⁴ with some modification. Briefly, monochromatic light was selected from six 40-W red fluorescent lamps in a custom-built projector using a red interference filter (λ=660 nm, 25 nm half bandwidth) (Roscolene No. 823 Kliegle Brothers, USA). The fluence rate was 10.5 Wm⁻² and was measured with Kettering Radiant Power Meter.

Protoplast volume measurement

The size of protoplasts was determined by photographing 100 μl protoplast suspension that was revolved at random by a disposable pipette from the treated population after an incubation period of 30 min in darkness at 20 ±2°C, and placed on a haemocytometer. At least 100 intact protoplasts were measured for each datum of the protoplast volume of each experiment.

⁴⁵Ca²⁺ transport assay

Accumulation of ⁴⁵Ca²⁺ in protoplasts was determined as described in the previous report¹⁵. 194 μl of protoplast suspension was incubated with 6 μl of ⁴⁵Ca²⁺ (74 MBq/ml), the reaction was stopped by adding 5 ml of cold washing solution containing 9% (w/v) mannitol, 5 mM MES and with or without 250 μM CaCl₂. The protoplasts were collected immediately on glass fiber filters and washed with 10 ml of fresh cold washing solution and counted in a G-M counter.

Protein content

The content of protein was determined by Folin-phenol assay.

Chemicals

Hemicellulase H-2125, pectinase, EGTA, verapamil, A₂₃₁₈₇, CPZ, TFP and Folin-B were purchased from Sigma Chemical Co., St. Louis, MO, USA. Cellulase Onzuka R-10 was purchased from Kinki Yakult Manuf. Co. Ltd. Japan. MES was purchased from Serva Feinbiochemica Heideberg, New York. ⁴⁵CaCl₂ was purchased from Institute of Atomic Energy, Chinese Academy of Sciences, Beijing. All other chemicals were analytical grade.

The data presented represent mean ± S.E. of three independent experiments. Statistical analysis was based on a one-way analysis of variance and significant differences at the 5% level were calculated.

Results

Effect of Ca²⁺ concentration on red-light-induced protoplast swelling and ⁴⁵Ca²⁺ accumulation in protoplasts

We have demonstrated previously that red-light-induced protoplast swelling occurred only when Ca²⁺ ions were present, other divalent (Ba²⁺, Mg²⁺ and Zn²⁺) and monovalent (K⁺) cations had no visible effects on protoplast swelling¹⁴. To investigate the effect of different external CaCl₂ concentration on swelling and ⁴⁵Ca²⁺ accumulation, protoplasts were suspended in the media containing 0 (plus EGTA), 100, 250, 500 and 1000 μM of CaCl₂, separately, and were irradiated with red light. Protoplast volume were determined after 30 min of incubation in darkness. As shown in Tab 1, both the volume and the accumulation of ⁴⁵Ca²⁺ of the protoplasts increased when CaCl₂ was present in the medium. The most effective concentration was 250 μM. the protoplast volume was 30.83% larger than that incubated in calcium-free control (+EGTA), while ⁴⁵Ca²⁺ accumulation was 61.09% higher than dark control in the same medium. Therefore this concentration was used in all subsequent experiments.

Tab. 1 The effect of different CaCl₂ concentration on red-light-induced protoplast swelling and ⁴⁵Ca²⁺ accumulation in protoplasts. Standard errors are shown for all data.

Ca²⁺ -dependence of protoplast swelling induced by red light

To investigate further the relationship between Ca²⁺ effect and the light signal, CaCl₂ (250 M) was added into calcium-free medium before or after red light treatment. It was shown that when the calcium was added 1 min after red light irradiation, swelling response decreased about 60% comparing to that added before light treatment. No significant swelling response occurred when CaCl₂ was supplied 5, 10, 20 min after red light irradiation (Fig 1).

Fig 1

The effect of Ca²⁺ addition before or after red light irradiation on red-lightinduced protoplast swelling. D (•); R(○). The final concentration of Ca²⁺ was 250 μM. Values and error bars were the mean and standard error of three replicate samples.

The specificity of Ca²⁺ on red-light-induced protoplast swelling

EGTA enter the cell membrane with difficulty, but it can chelate Ca²⁺ in the medium and specifically reduce the efficiency of external Ca²⁺¹⁷. When 0.25, 0.5 or 1 mM EGTA were added into Ca²⁺-containing medium, the red-light-induced protoplast swelling was inhibited. The stimulatory effect of red light was totally nullified at the concentration of 0.5 mM EGTA (Fig 2). In our experiments, EGTA at concentrations higher than 1.5 mM led to protoplast rupture.

Fig 2

The effect of EGTA on red-light- induced protoplast swelling. D(•); R(○).

Effect of verapamil on swelling and ⁴⁵Ca²⁺ uptake of protoplasts

Verapamil as a Ca²⁺-channel blocker can prevent selectively the influx of Ca²⁺ across the plasma membrane in some plant systems⁸. When 1, 5 and 10 μM verapamil were added, respectively, into the Ca²⁺-containing medium, ⁴⁵Ca²⁺ uptake was reduced and protoplast swelling was inhibited even though the protoplasts was irradiated with red light. At the concentration of 10 μM verapamil, 84.26% ⁴⁵Ca²⁺ uptake and 81.57% swelling response was inhibited (Fig 3).

Fig 3

The effect of verapamil on red-light-induced protoplast swelling and ⁴⁵Ca²⁺ accumulation in protoplasts. D (•, ♦); R (○, ⋄).

Effect of LaCl₃ on swelling and ⁴⁵Ca²⁺ uptake of protoplasts

La³⁺, an inorganic Ca²⁺-antagonist, can not enter plant cell, but it inhibits the movement of Ca²⁺ through the plasma membrane¹⁸. Treatment of the protoplasts with 10, 50 or 100 μM LaCl₃ did indeed inhibit ⁴⁵Ca²⁺ uptake and swelling response of protoplasts irradiated with red light in the Ca²⁺-containing medium. At the concentration of 100 μM of LaCl₃, 89.35% of ⁴⁵Ca²⁺ uptake and protoplast swelling was totally inhibited (Fig 4).

Fig 4

The effect of LaCl₃ on red-light-induced protoplast swelling and ⁴⁵Ca²⁺ accumulation in protoplasts. D (•, ♦); R (○, ⋄).

Effect of A₂₃₁₈₇ on swelling and ⁴⁵ Ca²⁺ uptake of protoplasts

A₂₃₁₈₇, a compound transporting Ca²⁺ across membrane down the concentration gradient¹⁹, was a kind of carboxylic antibiotic Ca²⁺ ionophore. In darkness, when 1, 5 or 10 μM of A₂₃₁₈₇ were supplied into the Ca²⁺-containing medium, ⁴⁵Ca²⁺ uptake was improved, and protoplasts swelled. The volume and ⁴⁵Ca²⁺ uptake in protoplasts were 82.91% and 90.62% ,respectively, as those of red light treatment when 5 μM of A₂₃₁₈₇ was added to medium (Fig 5).

Fig 5

Effect of A₂₃₁₈₇ on swelling and ⁴⁵Ca²⁺ uptake of protoplasts. D (•, ♦); R (○, ⋄).

The involvement of CaM in protoplast swelling

It has been shown that Ca²⁺ may act as secondary messenger, and combine with CaM in cytoplasm. The Ca²⁺-activated CaM then activates enzymes which regulates the physiological processes of plants³. To identify whether or not CaM involves in physiological modulation, CaM antagonists are generally used to test their inhibitory action. The previous results showed that treatment of protoplasts with 1, 5 or 10 μM of trifluoperizine (TFP) or chlorpromazine (CPZ), antagonists of the Ca²⁺-CaM complex, did indeed abolish the swelling of protoplasts irradiated with red light, and it was strongly blocked at the dose of 5 μM (Tab 2). Protoplasts rupture was observed at the concentration higher than 20 μM.

Tab. 2 Tab 2. The effect of TFP and CPZ on red-light-induced protoplast swelling. The concentration of CaCl₂ was 250 μM

Discussion

It has been demonstrated in plants that the environmental signals outside-cell are transduced to relative processes inside-cell via the change of Ca²⁺ concentration in cytosol. However, it is very significant but rather difficult to measure static or activated Ca²⁺ concentration in cytosol. So the participation of this well defined cellular signalling molecule in light signal transduction has been tested inside plant cells by using agonists or antagonists to examine their effects on light-mediated responses²⁰. Ca²⁺ influx into or efflux from plasma membrane or organ has been reviewed³. Hepler and Wayne⁵ showed that the changes of Ca²⁺ transport system meant the change of the concentration of intracellular Ca²⁺ when the spores of Onoclea were stimulated by red light. Recently, by means of laser scanning confocal microscopy, Fluo-3 was used to examine red-light-induced cytosolic free Ca²⁺ changes¹⁰. In our experiments, red-light-induced Ca²⁺ influx into protoplasts has been measured using ⁴⁵Ca²⁺. When CaCl₂ was at the concentration of 250-1000 μM, swelling response and the ⁴⁵Ca²⁺ uptake were positively correlated to concentration within certain limit, and the optimum concentration of external CaCl₂ on red-lightinduced swelling response of protoplasts was found to be 250 μM (Tab 1). The results demonstrated that Ca²⁺ was absolutely required for red-light-induced swelling of protoplasts. Moreover, full response was only observed when Ca²⁺ was added to the medium before red light treatment (Fig 1). Reduced response occurred when Ca²⁺ was added 1 min after light treatment and the swelling was totally prevented when Ca²⁺ was added to the calcium-free medium 5 min after red-light-irradiation, implying a pre-request of Ca²⁺ in red-light-induced swelling and this request is time dependent. Shacklock et al.¹⁰ reported that a transient rise in [Ca²⁺]_i lasting no more than 1 min was sufficient to elicit the prolonged physiological response of a change in cell volume.

If Ca²⁺ was chelated by EGTA, no protoplast swelling after red light-irradiation occurred (Fig 2). When the agents such as verapamil and LaCl₃ were used to block the Ca²⁺-channel, the red-light-induced ⁴⁵Ca²⁺ uptake and protoplast swelling were inhibited significantly. There was a good dose-relationship between percent of inhibition and concentrations of channel blockers (Fig 3 and 4). In darkness, A₂₃₁₈₇ could partially mimic red-light-induced swelling and promoting ⁴⁵Ca²⁺ influx in the presence of Ca²⁺ (Fig 5). All these facts indicates that protoplast swelling is a consequence of a rise in cytoplasmic Ca²⁺ concentration, and its inhibition by various inhibitors imply opening of Ca²⁺ channel stimulated by red light. Therefore our results support the hypothesis that red light induces an entrance of Ca²⁺ into protoplasts. Takagi and Nagai⁸ found that La³⁺ inhibit Ca²⁺ influx through Ca²⁺ channel in the cell membrane of protoplasts from Vallisneria mesophyll cell. Akerman et al²¹ observed that A₂₃₁₈₇ enhanced net Ca²⁺ accumulation by protoplasts. Mehta et a1²² suggested a role of ion channels and pumps in phytochrome-controlled Ca²⁺ fluxes and that modulation of [Ca²⁺]_i is likely to be achieved through changes in the activity of Ca²⁺ channels. These data suggested that dico and monoco plants share a common biochemical mechanism for at least part of protoplast swelling. Broad similarities between swelling of protoplasts induced by red light in mung bean and more well-studied monoco plants were apparent. However, there also existed considerable differences in detail, such as initial time, level and maintaining period of Ca²⁺ influx and protoplast swelling.

The participation of well-characterized cellular signalling molecules in light signal transduction has been tested as inside plant cells by using agonists or antagonists and by examining their effects on light-mediated responses²⁰. Ca²⁺ influx or efflux in plasma membranes or organs were studied³. The ability of TFP and CPZ to inhibit red-light-induced protoplast swelling (Tab 2) indicates that CaM might be involved in the transduction of light signal. When CaM is activated by Ca²⁺, it may then activate other target enzymes and physiological processes which result in the swelling of protoplasts. These drugs at higher concentrations may lead to disruption of protoplasts. In this case, their action is due to a non-specific disruption of membrane function rather than a specific CaM antagonism. These actions were highly similar to those found in monocotyledons. Similar phenomena have been noted by others²³.

In summary, this study provides a strong demonstration of calcium acting as a signaling molecule for phytochrome action in dico plants.