Carrot (Daucus carota L.) Protoplasts1 - NCBI

Plant Physiol. (1987) 83, 395-398 0032-0889/87/83/0395/04/$01.00/0

myo-Inositol Trisphosphate Mobilizes Calcium from Fusogenic Carrot (Daucus carota L.) Protoplasts1 Received for publication June 23, 1986 and in revised form October 3, 1986

MAGALY RINCON AND WENDY F. Boss*

Department of Botany, North Carolina State University, Raleigh, North Carolina 27695 ABSTRACI To determine whether or not inositol trisphosphate (IP3) mobilizes calcium in higher plant cells, we investigated the effect of IP3 on Ca' fluxes in fusogenic carrot (Ducas carota L.) protoplasts. The protoplasts were incubated in 'SCa2`containing medium and the 'Ca2 associated with the protoplasts was monitored with time. Addition of IP3 (20 micromolar) caused a 17% net loss of the accumulated ffCa2` within 4 minutes. There was a reuptake of 45Ca2@ and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP,), also stimulated 45"iC2 efflux from the protoplasts. Both the IP3- and the IP6-induced 4'(C2a efflux were inhibited by the calmodulin antagonist, trifluoperazine.

The polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, play a pivotal role in signal transduction in many animal cells in which agonists cause an increase in cytosolic calcium (1, 2, 8). Upon stimulation of these animal cells, the PIP22 located in the plasma membrane is hydrolyzed by a specific phosphodiesterase to DAG and IP3. Both DAG and IP3 may act as second messengers (for review see Berridge and Irvine [3]). DAG activates protein kinase C and is also used by the cells to resynthesize phosphatidylinositol (21). IP3 acts as a second messenger mobilizing calcium from intracellular stores (2, 14, 25). IP3 may be phosphorylated to form other inositol phosphates (19) or hydrolyzed in a 3-step dephosphorylation into inositol and Pi by specific phosphatases (3). Although very little is known about signal transduction in higher plant cells, calcium has become recognized as a key regulator of plant metabolism (11, 12, 18, 23, 26). It activates protein kinases (13), and plays a critical role in cell division (12), secretion (7), protoplast fusion (10), and tropic responses (22, 23). All of these physiological responses appear to be preceded by an increase of cytosolic free Ca2' concentration, but it is not clear whether the increase in cytosolic Ca24 concentration is due to an increase in Ca2` influx across the plasma membrane or to an increase in Ca2' release from internal stores (i.e. ER, vacuole, or mitochondria) or both. There is evidence that the biosynthesis and metabolism of polyphosphoinositides occur in higher plant cells (6, 20, 24). PIP and PIP2 have been identified in the wild carrot cells (Daucus carota L.) used in this study (6). In addition, these fusogenic 'Supported by the National Science Foundation (DCB-8502813). Paper No. 10829 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. 2Abbreviations: PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; IP6, myo-inositol hexaphosphate; IP3, myo-inositol trisphosphate; PIP, phosphatidylinositol 4-phosphate; TFP, trifluoperazine. 395

carrot cells have high rates of [3H]inositol incorporation into membrane lipids (5) and have been shown to closely regulate Ca2+ stores (10). Furthermore, PIP and PIP2 are localized in the plasma membrane fraction isolated from these cells by aqueous

polymer two-phase partitioning (27). If the metabolism of PIP2 is linked to the calcium signaling in plants via IP3, then increasing cytosolic IP3 should mobilize intracellular calcium. We report here that exogenous IP3 stimulates calcium efflux from fusogenic carrot protoplasts. The IP3induced calcium efflux is inhibited by the calmodulin antagonist TFP.

MATERIALS AND METHODS Protoplast Isolation. Wild carrot cells (Daucus carota L.) which yield fusogenic protoplasts, were grown in suspension culture as described previously (4). Protoplasts were isolated by placing 0.3 g of cells in 20 ml solution containing 2% Driselase (Plenum Scientific Co., Hackensack, NJ), 0.4 molal sorbitol, 2 mM EGTA, and 1 mm Mes (pH 4.8) at 25°C on a rotary shaker (125 rpm) for 1.5 h for the 45Ca24 efflux experiments and for 2 h for the 45Ca2+ influx experiments. The protoplasts were centrifuged (40g) and the supernatant discarded. The recovered protoplasts were washed twice with an osmoticum solution consisting of 0.45 molal sorbitol and 0.5 mM Mes (pH 6). 4"Ca2 Influx. For calcium influx experiments, protoplasts (12 mg protein/ml) were incubated in 8 ml osmoticum containing 45Ca2" (0.4 MCi/ml) to a final Ca2" concentration of approximately 1 uM. The protoplast suspension was placed on the rotary shaker (90 rpm) at 25°C. At intervals, 3 aliquots of 200 ,l were taken and protoplasts spun in a Beckman Microfuge B through a supporting and separating gradient formed, in order from the bottom, by 50 Ml of 5% dextran (mol wt = 17,900) in osmoticum, 50 ,ul silicone oil (p = 1.03), 50 ,l 2 mM EGTA in osmoticum, and 50 gl silicone oil (p = 1.01). The protoplast pellet was collected at the bottom of the microfuge tubes. The microfuge tips were cut and the protoplast pellet resuspended in 300 Ml deionized H20. Aliquots of 100 ,l ofthe resuspended protoplasts were dissolved in 7 ml Scintiverse-Il (Fisher). The radioactivity was determined by liquid scintillation counting in a Beckman LS 7500 scintillation system. Protein was assayed as described by Lowry et al. (17). 45Ca24 Efflux. For calcium efflux experiments, protoplasts (12 mg protein/ml) were incubated for 30 min in 10 ml osmoticum containing 45Ca2` (5 MCi/ml) to a final Ca2' concentration of approximately 5 gM. The radioactive solution was centrifuged and the supernatant discarded. Protoplasts were washed with 1.5 ml of osmoticum containing 2.26 mm EGTA, centrifuged and washed twice with osmoticum. The 45Ca2+ labeled protoplasts were resuspended in 6 ml of osmoticum and placed on the rotary shaker (90 rpm). To follow the 45Ca2+ loss from the protoplasts, aliquots were withdrawn and the 45Ca2` content determined as described above, with the exception that the 2 mm EGTA in

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osmoticum and the top silicone layers were omitted. When the effect of TFP on the 45Ca2" efflux was investigated, TFP ( 15 AM) was added during the last 30 min of the cell wall digestion, as well as during the 45Ca2" loading and the 45Ca2" effiux. Stock Solutions. Stock solutions (3 mM) of IP6 and IP3 were prepared in the osmoticum and used immediately or stored up to 2 weeks at -20°C. A stock solution of TFP (4 mM) was made in deionized H20. The calcium ionophore, A23 187 (2 mM), was prepared in absolute ethanol. All chemicals were purchased from Sigma except for the calcium ionophore, A23187, which was purchased from Calbiochem and 45Ca2+ which was purchased from Amersham. RESULTS If in plant cells PIP2 is hydrolyzed in response to external stimuli and the IP3 formed releases Ca2+ from internal stores, then increasing the cytosolic IP3 by adding IP3 (in the absence of a stimulus) should mobilize intracellular Ca2+. The subsequent increase in cytosolic free Ca2' could activate Ca2+ pumps associated with the plasma membrane and result in a net Ca2' efflux from the cells. To test this hypothesis, we investigated the effect of IP3 on 45Ca2+ fluxes in fusogenic carrot protoplasts. The time course of 45Ca2+ influx by the carrot protoplasts and the effect of IP3 are illustrated in Figure 1. There was a 17% loss of the 45Ca2+ accumulated by the protoplasts within 4 min after 20 ,LM IP3 addition. The protoplasts recovered their previous 45Ca2+ content within 10 min; however, the recovery period was variable and in one instance, the protoplasts did not regain their initial levels of 45Ca2+ by the time the experiments were terminated. When 5 ,uM IP3 was used, no change in the 45Ca2+ fluxes was observed; however, 20 AM IP3 gave a consistent response and, therefore, this concentration was used throughout this study. While plasma membranes are not readily permeable to IP3, the changes in the 45Ca2+ levels of the protoplasts observed upon IP3 addition suggested that the carrot protoplast plasma membrane was already permeable to IP3. This may be due to the fact that the carrot cells had been exposed to a crude mixture of fungal enzymes containing cellulases and proteases. Thus, the cell wall digestion process may have rendered the protoplast plasma membrane permeable to IP3. In addition, attempts to increase the permeability of the protoplast plasma membrane to IP3 were pursued. Saponin treatment (50 ,ug/ml) for 4 min, commonly used to permeabilize animal cells to IP3 (2, 14), was

Plant Physiol. Vol. 83, 1987

found to be ineffective with the carrot protoplasts; that is, there was no IP3 response (data not shown). Furthermore, if the cell wall digestion was carried out in the presence of 2 mM EGTA, the response to IP3 was more pronounced. For example, in one experiment upon IP3 addition, the EGTA-treated protoplasts lost 14% of the accumulated 45Ca2+ within 2 min, whereas protoplasts isolated in the absence of EGTA only lost 9%. The EGTA treatment did not affect protoplast viability (15) and no measurable loss of protein was evident during the course of the 45Ca2+ influx. Thus, 2 mM EGTA was added during the cell wall digestion throughout this study. To further support the thesis that the transient loss of 45Ca2" observed in the uptake experiments was due to an efflux and not to a reduced influx, 45Ca2" efflux experiments were conducted. The time course of 45Ca2" efflux from the protoplasts is illustrated in Figure 2. In this experiment, 45Ca2+-preloaded protoplasts were placed in Ca2+-free medium and the 45Ca2' remaining in the protoplasts was determined. IP3 enhanced the 45Ca2+ efflux from the protoplasts. These data suggested that the 45Ca2+ released from internal stores by IP3 was being pumped out of the protoplasts. Since IP3 mobilized Ca2+ from the protoplasts, the question arose as to whether or not the response was calmodulin regulated. To address this question, the protoplasts were pretreated with 15 ,gM TFP during the last 30 min of the cell wall digestion, as well as during the 45Ca2+ loading of the protoplasts and the efflux experiment. As shown in Figure 3, the IP3-induced 45Ca2+ efflux was affected by the calmodulin antagonist, TFP. TFP not only eliminated the IP3-induced 45Ca2+ efflux, but also caused a decrease in the 45Ca2+ levels of the protoplasts. IP6, a potential precursor of IP3, also affected the 45Ca2+ fluxes of these protoplasts (Fig. 4). The response to IP6 was similar to that of IP3; however, the protoplasts were never observed to recover their original 45Ca2+ level by the time the experiments were terminated. The effect of the calmodulin antagonist, TFP, on the IP6-induced 45Ca2+ efflux was also investigated. TFP (10 Mm) was present only during the 45Ca2+ uptake and as shown in Figure 4, TFP blocked the IP6-induced 45Ca2' efflux. 0

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FIG. 1. Effect of IP3 on the 45Ca2+ influx of the fusogenic carrot protoplasts. IP3 (20 AM) was added at the indicated time. Each point is the mean ±SD of three replicates. Standard deviation bars are not drawn when smaller than the symbols. Similar results were obtained in three separate experiments.

FIG. 2. Effect of IP3 on the 45Ca2" efflux from the fusogenic carrot protoplasts. The protoplasts were preloaded with 45Ca2" and the excess label was removed by washing the protoplasts with osmoticum containing EGTA as described in "Materials and Methods." The protoplasts were placed in Ca2"-free osmoticum, and 45Ca2" remaining in the protoplasts determined. IP3 (20 gM) was added at the indicated time; an equivalent volume of osmoticum was added to the control. Each point is the mean +SD of three replicates. Similar results were obtained in four separate experiments.

myo-INOSITOL TRISPHOSPHATE AND Ca2" FLUXES

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FIG. 3. Effect of TFP on the IP3-induced 45Ca2" efflux. The protoplasts were treated with 15 gM TFP as described. The protoplasts were allowed to efflux 45Ca`+ prior to additions (U). IP3 (20 AiM) was added at the indicated time (--); an equivalent volume of osmoticum was added to the control (0). The calcium ionophore, A23187 (10 Mm), was added to both IP3-treated and control protoplasts at the indicated time. Each point is the mean of three replicates. Similar results were obtained in two separate experiments.

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FIG. 4. Effect of 1P6 on the 45Ca2+ influx of the fusogenic protoplasts.

IP6 (10 MM) was added at the indicated time. TFP (10 ,uM) was added 10 min prior to 45Ca2+ labeling. Each point is the mean +SD of three replicates. Similar results were obtained in two separate experiments.

DISCUSSION The data indicated that 1P3 induced a transient effux of Ca2+ from the fusogenic carrot protoplasts (Figs. 1 and 2). This effux might be due to a stimulation of Ca2+ pumping mechanisms by the increased cytosolic free Ca2+. The percentage of 45Ca2+ lost from the protoplasts was small when compared to the 50% loss induced by 1P3 in Swiss mouse 3T3 cells (2) and was more comparable to the 25% loss of calcium observed in the 1P3treated peritoneal macrophages (14). The magnitude ofthe "5Ca2+ efflux observed in the carrot protoplasts might be due to the fact that plant cells, unlike animal cells, have a large reservoir for intracellular calcium, the vacuole. The tonoplast contains an inward-directed, ATP-dependent calcium pump which, if activated by the increased cytosolic free calcium, would decrease the net efflux of calcium from the protoplasts. Thus, the 1P3-induced effux would be expected to vary depending upon the capacity of the vacuole to sequester calcium (i.e. the larger the vacuole, the less the observed efflux and the greater the potential for

FIG. 5. Diagram summarizing a possible role of IP3 in the stimulusresponse mechanism in higher plant cells. External stimuli activate the plasma membrane PIP2 phosphodiesterase, and as a result, PIP2 is cleaved to DAG and IP3. IP3 serves as second messenger mobilizing calcium from internal stores. ER, endoplasmic reticulum; V, vacuole; PDE, phosphodiesterase; CaM, calmodulin; IP2, myo-inositol bisphosphate; IP, myo-inositol monophosphate.

calcium to be pumped into the vacuole). The magnitude and the rate of calcium efflux would also depend upon the permeability of the plasma membrane toward IP3 and the metabolism of IP3 by the protoplasts. The transient nature of the response to IP3 reflected the ability of the protoplasts to metabolize inositol phosphates (cf. Figs. 1 and 4). This was further exemplified by the sustained response to IP6, which suggested continued metabolism of IP6 to an active isomer since phytases and other phosphatases are prevalent in plants (16). The response was not due to nonspecific binding of calcium by these compounds since neither IP3 nor IP6 affected the levels of the 45Ca2' associated with the saponin-treated protoplasts (data not shown). In addition, IP3 and IP6 did not stimulate calcium mobilization in the TFP-treated protoplasts. Furthermore, it should be noted that the IP3 and IP6 treatments affected 45Ca2+ fluxes due to the fact that the externally bound 45Ca2+ was removed by the 2 mM EGTA present in the osmoticum layer above the silicone oil gradient. Other factors which are important in assessing the response of the protoplasts to IP3 include the purity and stability of the purportedly active isomer involved in the mobilization of cal-

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cium, inositol 1,4,5-trisphosphate. IP3 is not stable in acidic aqueous solutions and, therefore, it would be unstable in the weakly buffered solutions (pH 6) used for these studies. In addition, as we suggested earlier, IP3 might be metabolized by the carrot protoplasts as in animal cells (3, 19). Thus, the relatively high concentration of IP3 (20 ,uM) needed for these studies, as compared to the 1 to 5 ,uM used for a maximum response in animal cells (2, 14), might reflect differences in the purity of the starting material and/or differences in cellular metabolism and the permeability of the plasma membrane to IP3. The role for a Ca2+-calmodulin regulated Ca2" pump associated with the plasma membrane was implied by the effect of the calmodulin antagonist, TFP, on the 45Ca2+ fluxes. When the protoplasts were treated with TFP, there was no longer an IP3stimulated efflux even though the calcium ionophore, A23187, caused an efflux of 45Ca2" (Fig. 3). It is not clear from these in vivo experiments whether TFP affected the sequestration of 45Ca2" into the IP3-sensitive store or affected the Ca2" pump at the plasma membrane. Although relatively low concentrations of TFP (10 and 15 gM) were used in these studies, one must be cautious in interpreting these results due to the potential for nonspecific effects of the phenothiazine drugs on the membranes. While the source of IP3-mobilized calcium in the carrot cells is as yet unidentified, a nonmitochondrial store appears to be the site of IP3 action in plant cells, as suggested by the results of Dr0bak and Ferguson (9). They found that IP3 stimulated release of accumulated calcium from a crude microsomal fraction isolated from zucchini hypocotyls. The potential role for IP3 in stimulus-response coupling in plant cells is depicted in Figure 5. The IP3 would stimulate the release of calcium from internal stores such as the ER, and the subsequent increase of cytosolic calcium could regulate cellular metabolism by activating enzymes (e.g. protein kinases) and other calcium/calmodulin-regulated responses. The diagram also illustrates the Ca2"-pumping mechanisms which may also be activated to lower the cytosolic free calcium to the resting levels. Studies are in progress to determine which store(s) is the site of IP3 action in the fusogenic carrot cells and whether or not the vacuole plays a role in mediating the IP3-induced calcium mobilization. Acknowledgments-The authors would like to acknowledge Mara Massel and Mr. Scott Nally for their technical assistance. LITERATURE CITED 1. BERRIDGE MJ 1983 Rapid accumulation of inositol trisphosphate reveals that

agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 212: 849-858 2. BERRIDGE MJ, JP HESLOP, RF IRVINE, KD BROWN 1984 Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to plateletderived growth factor. Biochem J 222: 195-201 3. BERRIDGE MJ, RF IRVINE 1984 Inositol trisphosphate, a novel second messen-

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ger in cellular signal transduction. Nature 312: 315-321 4. Boss WF, HD GRIMES, A BRIGHTMAN 1984 Calcium-induced fusion of fusogenic wild carrot protoplasts. Protoplasma 120: 209-215 5. Boss WF, HD GRIMES 1985 Dynamics of calcium-induced fusion of fusogenic carrot protoplasts. In JB St John, E Berlin, PC Jackson, eds, Frontiers of Membrane Research in Agriculture, Vol 9. Rowman & Allanheld Publishers, Totowa, NJ, pp 133-145 6. Boss WF, M MASSEL 1985 Polyphosphoinositides are present in plant tissue culture cells. Biochem Biophys Res Commun 132: 1018-1023 7. CARBONELL J, RL JONES 1985 A comparison of the effects of Ca2" and gibberelic acid on enzyme synthesis and secretion in barley aleurone. Physiol Plant 63: 345-350 8. CREBA JA, CP DOWNES, PT HAWKINS, G BREWSTER, RH MICHELL, CJ KIRK 1983 Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones. Biochem J 212: 733-747 9. DR0BAK BK, IB FERGUSON 1985 Release of Ca2" from plant hypocotyl microsomes by inositol-l ,4,5-trisphosphate. Biochem Biophys Res Commun 130: 1241-1246 10. GRIMES HD, WF Boss 1985 Intracellular calcium and calmodulin involvement in protoplast fusion. Plant Physiol 79: 253-258 11. HANSON JB 1984 The functions of calcium in plant nutrition. In PB Tinker, A LAuchli, eds, Advances in Plant Nutrition, Vol 1. Praeger Publishers, New York, pp 149-208 12. HEPLER PK, RO WAYNE 1985 Calcium and plant development. Annu Rev Plant Physiol 36: 397-439 13. HETHERINGTON AM, AJ TREWAVAS 1984 Activation of a pea membrane protein kinase by calcium ions. Planta 161: 409-417 14. HIRATA M, E SUEMATSU, T HASHIMOTO, T HAMACHI, T KOGA 1984 Release of Ca2" from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate. Biochem J 223: 229236 15. KANCHANAPOON K, WF Boss 1986 The effect of fluorescent labeling on calcium-induced fusion of fusogenic carrot protoplasts. Plant Cell Rep. 861:

429-439 16. LOEWUS FA, MW LOEWUS 1983 myo-Inositol: its biosynthesisand metabolism. Annu Rev Plant Physiol 34: 137-161 17. LOWRY DH, J ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with Folin reagent. J Biol Chem 193: 265-275 18. MARME D 1983 Calcium transport and functions. In A Lauchli, RL Bieleski, eds, Encyclopedia of Plant Physiology, Vol 15. Springer-Verlag, Heidelberg, pp 599-625 19. MICHELL B 1986 Inositol phosphates. Profusion and confusion. Nature 319: 176-177 20. MORSE MJ, RC CRAIN, RL SATTER 1986 Phosphatidylinositol turnover in Samanea pulvini: a mechanism of phototransduction. Plant Physiol 80: S92 21. NISHIZUKA Y 1984 Turnover ofinositol phospholipids and signal transduction. Science 225: 1365-1370 22. PICKARD BG 1985 Roles of hormones, protons, and calcium in geotropism. In RP Pharis, DM Reid, eds, Encyclopedia of Plant Physiology, Vol 11. Springer-Verlag, Berlin, pp 193-281 23. Roux SJ, RD SLOCUM 1982 Role of calcium in mediating cellular functions important for growth and development in higher plants. In WY Cheung, ed, Calcium and Cell Function, Vol 3. Academic Press, New York, pp 409-453 24. SANDELIUS A, M SOMMARIN 1986 Phosphorylation of phosphatidylinositol in isolated membranes. FEBS Lett. 201: 282-286 25. STREB H, RF IRVINE, MJ BERRIDGE, I SCHULZ 1983 Release of Ca2" from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol1,4,5-trisphosphate. Nature 306: 67-68 26. TREWAVAS AJ, R SEXTON, P KELLY 1984 Polarity, calcium and abscission: molecular basis fordevelopmental plasticity in plants. J Embryol Exp Morph 83: 179-195 27. WHEELER JJ, WF Boss 1986 Are polyphosphoinositides in the plasma membrane of carrot cells? Plant Physiol 80: S-80