Measuring cytoplasmic calcium level in Citrus protoplasts using the fluorescent probe indo-1

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Measuring cytoplasmic calcium level in Citrus protoplasts using the fluorescent probe indo-1 CIBELE 1

M. C. P. GouvtA\

BENEDITO

c. VIDAL2, and lONE S. MARTINS l *

Departamento de Bioqufmica and, 2 Departamento de Biologia Cellular, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, Campinas, 13083-970, Brazil

Received August 12, 1996· Accepted November 30, 1996

Summary

We measured cytoplasmic free calcium in protoplasts of Citrus sinensis Osb. Protoplasts were isolated from cell-suspension cultures grown as microcalli. The cell surface presented acid polysaccharide filaments, probably responsible for cell adhesion. These filaments were disrupted by enzymatic digestion liberating the cells and subsequently the protoplasts. Protoplasts were loaded with the pentapotassium salt of indo-l by incubation in acidic solutions of this calcium indicator. Loading increased with decreasing pH and osmotica. Dye loaded in this manner was distributed only in the cytoplasm. The protoplast autofluorescence was very low and did not interfere with measurements. Cytoplasmic free-calcium concentration in Citrus protoplasts, using microspectrofluorimetry, was estimated to be 154 nmol . L-I. This resting value was increased when protoplasts were exposed to high extracellular Ca2 + (1 mmol . L-1) or 6BA (1 J.lmol· L-I) suggesting that the pathway in cytokinin signal transduction in these cells may involve a Ca2 + uptake facilitation from extracellular medium.

Key words: Citrus sinensis Osb., cytokinin, cytosolic calcium, indo-I, protoplast. Abbreviations: indo-l = 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy] -2-(2'-amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid, pentapotassium; 6BA =6-benzylaminopurine.

Introduction

Calcium is an essential nutrient element in plants, playing its physiological and biochemical roles in the apoplast, on the plasma membrane, and in the cytoplasm. The central importance of ea2 + signaling in plants is now well established (Hepler and Wayne, 1985; Trewavas and Gilroy, 1991; Bush, 1995). Its regulatory actions range from control of ion transpon to gene expression and are possible because of a homeostatic system that regulates Ca2 + levels. These regulatory propenies depend largely on the maintenance of low calcium levels in the cytoplasm, which are modulated by coordinating passive fluxes and active transpon across the plasma and * Correspondence. j. Plant PhysioL WJL 151. pp. 329-333 (1997)

organellar membranes. Increases in cytoplasmic Ca2 + levels activate Ca2 +-dependent proteins, stimulate protein phosphorylation, and lead to initiation of diverse cellular processes (Read et al., 1992; Roberts and Harmon, 1992; Poovaiah and Reddy, 1993; Bush, 1993; 1995). Calcium has been proposed as a major regulator of signal transduction in plant cells in response to hormones (Hepler and Wayne, 1985; Bush, 1996). Extensive work has been done on cytokinin-calcium interaction in plants such as moss, and it has been established that cytokinins induce an asymmetrical cell division in those plant cells. The mechanism leading to these cytological rearrangements is not well understood, but a spatially- and temporally-controlled rise in intracellular Ca2 + has been implicated as pan of cytokinin action (Schumaker and Gizinski, 1993). Additional lines of

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CIBELB M. c. P. GouvtA. BENEDITO C. VIDAL. and lONE S. MAImNS

evidence suggest that it is the Ca2+ uptake from the extracellular medium that mediates the cellular response to cytokinins (Hahm and Saunders. 1991). A desire to understand the role of Ca2 + in plant signal transduction has oriented much of the recent research toward regulation of cytosolic Ca2 +. A crucial step has been the development of techniques for measuring the cytosolic calcium (Read et al.• 1992). Methods to measure cytosolic Ca2 + developed for animal cells can not always be directly applicable to plant cells because of their structure. Despite such problems. a number of techniques for measwing cytoplasmic Ca2+ have now been successfully applied to plants. but only in a narrow range of cell types (Read et al .• 1992; Bush, 1995). We focused our attention on the Ca2+ -sensitive fluorescent dye indo-l which exhibits higher affinity and selectivity for calcium ions than other probes, and its dual emission wavelength property allows the precise quantitation of free Ca2+ concentration (Grynkiewicz et al., 1985; Bush and Jones, 1987). Its utility for measuring cytosolic Ca2 + levels depends on the dye remaining in the cytoplasm and not being bound or sequestered in other cellular compartments. In the present study we obtained Citrus suspension-cultured cells, isolated viable protoplasts, optimized the technique for acid loading plant protoplasts with indo-l (Bush and Jones, 1987), and measured the effect of 6BA on the resting level of cytoplasmic calcium.

Materials and Methods

Plant 1'1Ulteri41 Suspension-cultured cells of Citrus sinmsis Osb. cv. Pera were established from nucellar calli as described by O'Utra Vaz et al. (1993). The cells were maintained in liquid nutrient medium of Murashige and Skoog (1962) containing vitamins (Murashige and Tucker, 1969), 50 g. L-1 sucrose. 0.1 g. L-1 myoinositol. 0.5 g. L-I malt extract and 10 mg· L-1 6BA The culture was kept in the dark. at 26 ± 2 ·C with constant shaking (150 rev. min-I) and subcultured every 14d.

Protoplast isolatWn Protoplasts were isolated from suspension-cultured cells of Citrus according to O'Utra Vaz et al. (1993). The cells were placed in CPW (Frearson et al .• 1973) containing 130 g. L-1 mannitol (CPW-mannitol) for plasmolysis (1 h). The CPW-mannitol solution was replaced by an enzyme mixture containing 15 g. L-1 cellulase, 2 g. L-1 hemicellulase and 20 g . L-1 pectinase in CPW-mannitol solution (pH 5.6). The material was incubated for 16 h with constant shaking (40 rev. min-I). at 26 ± 2 ·C, in the dark. Protoplasts were separated from undigested tissue by filtering through a nylon sieve (45 11m pore size). Protoplasts were pelleted and washed three times in CPW-mannitol with centrifugation at 100 g., (4 min) between each wash. The pellet was resuspended in CPW-mannitol and passed through CPW containing 250 g . L-1 sucrose and centrifuged at 100 g., (8 min). After centrifugation. protoplasts at the surface were collected and washed three times in CPW-mannitol. Protoplasts were counted and then viability assessed using Trypan blue (O.lg . L- I).

Microscopy Phase contrast microscopy was carried out to identify the cell and protoplast morphology and to control the fluorescence measurements. Polarization microscopy was used to detect optical anisotropy of cell components.

Indo-J loading Protoplasts were loaded with the pentapotassium salt of indo-1 according to Bush and Jones (1987) with modifications. Protoplasts were suspended in 20 mmol· L-1 dimethylglutaric acid pH 4.5 containing 0.52 mol· L-1 mannitol. 50 mmol . L-1 KCI and 20 I1mol . L- 1 indo-I. After 2 h incubation at room temperature. protoplasts were washed twice and resusgended in 20 mmol · L-1 Tris-HCI pH 7.2. containing 0.52 mol· L- mannitol and 50 mmol· L-1 KCl. The resulting protoplasts showed the ability to exclude ~rypan blue and to accumulate neutral red. both stains at 0.1 g. L-I, thus exhibiting high intactness. FlUQrescmc~

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Calcium-dependent indo-1 fluorescence from individual protoplasts was measured with a Zeiss microspectrophotometer equipped to perform microfluorimetry with the III RS condenser. as described by Mello and Vidal (1985). Operating conditions were: 0.250 mm measuring diaphragm diameter positioned in front of a monochromator ruler, 365/6 nm exciting light. FT 420 chromatic splitter. LP 410 and 480 barrier filter. An HBO-100W stabilized mercury lamp was employed as light source, and an HTV-R 446 photomultiplier was also used. Arbitrary fluorescence intensity of individual protoplasts was recorded. and the cytoplasmic calcium concentration was estimated using the ratio technique of Grynkiewicz et al. (1985). R min and R max values were determined from the measurement of fluorescence intensities of incubation medium either with 10 mmol . L-1 EGTA or saturating CaCl 2 (4 mmol· L-1) . We also used buffered Ca2+ solutions.

Results and Discussion

The microscopic analysis of the Citrus cell-suspension cultures showed that the cells grow as microcalli, some of them forming tightly packed cell masses that have a thick and birefringent cell wall, dense cytoplasm. and very low autofluorescence intensity (Figs. 1A; 1 C). The staining with toluidine blue pH 4.0 revealed metachromasy on the cell wall, in the cytoplasm, and in the filaments sprouting from the cell wall (Fig. 1 B). This type of metachromatic basophily is compatible with the presence of polygalacturonic acid (pectin). The filaments of the cell surface are likely to be responsible for cell adhesion because when they were digested with pectinase the cells became separated. Protoplasts isolated from those cells showed different sizes, a conspicuous nucleus and vacuole, and several cytoplasmic granules that appeared to have molecular organization and were formed by starch (Figs. 2 A; 2 B). The protoplast autofluorescence was very low and did not interfere with the emission wavelength ofindo-l-Ca2 +. The pH-method to load indo-l (Bush and Jones, 1987) used for some plant cells was only successfull a&er the modifications introduced, as described in «Material and Methods». We observed a significant increase in the loading of indo-I as a function of decreasing pH and osmotica. The pat-

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Fag. 1: Suspension culture of Citrus sinensis (L) Osb, -cv, Pera, A, The cells grow as mictocalluses, forming tightly packed cell masses. Arrows indicate filaments on the cell surface, probably responsible for cell adhesion. B, Cells treated with toluidine blue pH 4.0. Arrows indicate the presence of acid polysaccharide in the filaments (F), cell wall (CW) and cytoplasm (C). C, Birefringent images of the cells. The cell wall (CW) is thick and birefringent with a secondary cell wall (S). The cytoplasmic granules are formed by starch (arrow). Bar = 50j.1m.

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tern of cytoplasmic fluorescence of the cells loaded in this manner was diffuse, and no sign of vacuolar fluorescence was detectable (Figs. 2 C; 2 D). Our results were similar to those reported earlier by Bush and Jones (1987) for barley aleurone protoplasts. The conditions necessary for measuring internal [Ca2+] in indo-I-loaded protoplasts were found to be fulfilled: (i) indo-l does not leak out of the cells when they are immersed in a buffer of pH 4.5; (ii) indo-l accumulates only in the cytoplasm; and (iii) intracellular indo-l is not modified by cellular metabolism. Cytoplasmic calcium concentration in Citrus protoplasts was estimated to be 154.04 ± 25.02 nmol· L-1 (Fig. 3). This mean value resulted from 5 different protoplast preparations with 20-30 individual measurements in each. The statistical analysis (One-Way ANOYA) showed that the mean values of free [Ca2 +] determined for these populations were not significandy different at the 0.05 level. The internal [Ca2+] of isolated protoplasts of Citrus is well within the range of cytosolic [Ca2+] determined for cells from other plant species using indo-I, such as 250 nmol . L-1 in barler aleurone protoplasts (Bush and Jones, 1987), 93 nmol· L- in maize protoplasts (Lynch et al., 1989), 376 nmol· L-1 in yeast cells (Halachmi and Eilam, 1989), 113 nmol· L-1 in Amaranthus tricolor protoplasts (Elliott and Petkoff, 1990), 257 nmol· L-1 in protoplasts from the maize root elongation wne and 160 nmol. L-1 from the root cap (Kiss et al., 1991), 250 nmol· L-1 in Punaria (Hahm and Saunders, 1991), and 200 nmol. L-1 in Vida foba guard-cell protoplasts (Darjania et al., 1993). When Citrus protoplasts were exposed to 1 mmol· L-1 extracellular calcium, the concentration of cytoplasmic Ca2+ raised from 154 to 246 nmol· L-1 (Fig. 4). This rise in [Ca2+] probably resulted from import of calcium from the medium since it was evoked by increasing the electrochemical potential of Ca2+ across the plasma membrane. Increases in cytoplasmic Ca2+ following exposition to high concentrations of extracellular calcium have also been described to occur in barley aleurone protoplasts (Bush and Jones, 1988) and root hairs of Zea mays (Felle, 1988). The Citrus cells line used in this work requires exogenous supply of 6BA to maintain high rates of growth in culture. As alteration in calcium homeostasis has been considered part of the cytokinin signal transduction pathway (see Gilroy et al., 1993; Bush, 1995), we analysed the effect of 6BA on the cytoplasmic [Ca2+] of the Citrus protoplasts isolated from cytokinin susceptible cells. Cytoplasmic [Ca2 +] increased approximately twice when Citrus protoplasts were incubated with 1 J1mol. L-1 6BA (Fig. 4). The value of cytoplasmic [Ca2 +] following 6BA treatment (218 nmol· L-1) was further increased to 320 nmol· L-1 in the presence of 1 mmol. L-1 calcium, what was observed in every cell measured. These data indicate that the facilitation of calcium influx through the plasma membrane by increasing the electrochemical gradient for Ca2+ is potentiated by 6BA. Cytokinin has also stimulated calcium influx into the protoplasts from cotyledons of Amaranthus tricolor (Elliot and Yuguang, 1989), but in response to a much higher level of 6BA (I mmol· L-1). In moss protoplasts, 0.1 J1mol. L-1 6BA has caused a stimulation of calcium influx without prior incubation of the protoplasts with the phytohormone (Schumaker and Gizinski, 1993). These authors proposed that one of the cytokinin effects

332

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Fig. 3: Frequence distribution of cytoplasmic calcium levels in resting Citrus protoplasts loaded with indo-I. The mean value of cytoplasmic calcium concentration was estimated to be 154.04 ± 25.02 nmol. L-1.

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Treatments: 1, control; 2, 1 mmol. L-1 ea +; 3, Il1mol. L-1 6BA; 4, Il1mol. L-1 6 BA + 1 mmol· L-1 Ca2+; 5, Il1mol. L-1 adenine. Mean values of at least 50 determinations, after 10 min incubation in each reatment.

FJgo 21 Freshly isolated protoplasts &om suspension-cultured cells of Citrus sinensis (L.) Osb. cv. Pera. A, D, Bire&ingent image. The cytoplasmic granules are formed by starch (arrow). Bar = 50l.lm. C, D, Fluorescent images of protoplasts loaded with 20 I1mol. L-1 pentapotassium salt of indo-I. The fluorescence is restricted to the cytoplasm, Bar = 100 11m,

support the proposal that the cytokinin siynal transduction pathway may involve the opening of a Ca + channel at the plasma membrane. Calcium channels have been shown to be involved in the mediation of early events of many different signal transduction pathways in plant cells (Ward et al.,

would be the regulation of a plasma membrane voltagedependent dihydropyridine-sensitive ci+ channel. Our results on the 6BA-induced increase in cytoplasmic [Ca2 +]

The free base adenine had no effect on the cytoplasmic [Ca2 +] of Citrus protoplasts (Fig. 4) indicating that the observed response to 6BA was not due to some non-specific perturbation of the cells.

1995).

Cytoplasmic calcium level in Citrus protoplasts

Although calcium is known to be fundamental in a lot of developmental processes the maintenance of high intracellular calcium can lead the cell to death (Clapham, 1995). In the recent literature concerning the role of calcium signalling in plant cells, many different stimulus have been shown to induce an increase in the cytoplasmic [Ca2 +]. Although in some cases the observed rise is large, there are a lot of examples where minor increases in cytoplasmic [Ca2+] induced by different stimuli are enough to promote the physiological response (see Gilroy et al., 1993). It seems as though, in Citrus cells slight increases in cytoplasmic [Ca2+] evoked by 6BA may be enough to activate the overall metabolism and have significance on the control of cell division. Acknowledgements

CMCPG was supported by a pos-doctoral fellowship from Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq). This research was supported by a grant from Fundacrao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP). We are grateful to Pro£ Fernando D'Utra Vaz from the Centro de Energia Nuclear na Agricultura - Universidade de Sao Paulo (CENA-USP) for generously providing the initial Citrus cell culture.

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