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Do polyamines release membrane-bound calcium in sugar beet protoplasts?
..........,.....,
• J••••AL.F.
Short Communication
© 1998 by Gustav Fischer Verlag. Jena
Do Polyamines Release Membrane-bound Calcium in Sugar Beet Protoplasts? ANNA MAJEWS~-SAWKA,
RAFAI. BUTOWT, and ALEKSANDRA NU(LAs*
Institute for Plant Breeding and Acclimatization, Powstaflc6w Wielkopolskich 10, 85-950 Bydgoszcz, Poland
* Permanent address: University of Technology and Agriculture, Kaliskiego 7, 85-791 Bydgoszcz, Poland Received March 17, 1997 . Accepted August 26, 1997
Summary The possible effect of exogenous polyamines on the changes in calcium ion content in sugar beet protoplasts maintained in calcium-free media was studied. We combined the chlorotetracycline fluorescent method (CTC) with direct measurements of Ca2+ levels in a Magical system. St<=rmine and spermidine at concentrations of 50, ,100 and 300 J.UI1ollL caused a conspicuous increase of Ca + detectable in the vicinity of membranes, whereas putrescine had no such effect. The specificity of fluorescence for CTC-Ca complex was confirmed by EGTA treatment, and the membranous origin of fluorescence was confirmed by the fact that it was abolished by Triton X-100. Polyamines are therefore postulated to act by displacing Ca2 + from intracellular membranes.
Key words: Beta vulgaris L., calcium, polyamines, protoplasts. Abbreviations: CTC = chlorotetracycline; EGTA = ethylene glycol-bis-(-aminoethylether)- N,N,N',N'tetraacetic acid; mMS = modified Murashige and Skoog medium; Put = putrescine; Spd = spermidine; Spm spermine.
=
Introduction Polyamines affect a wide range of physiological functions in plant cells, including synthesis of RNA, DNA and proteins, stabilization of plasma membranes and transduction of extracellular signals (liburcio et al., 1993). The cellular mechanisms by which these compounds act have been studied extensively in many systems in vitro, and protoplasts are an especially attractive model for experiments with exogenous polyamines (Antognoni et al., 1995). Exogenous spermine and spermidine are rapidly ttanspotted into the protoplasts through a carrier-mediated mechanism (Antognoni et al., 1994), and may stimulate the protoplast division (KaurSawhney et al., 1980; Eilers et al., 1988; Majewska-Sawka et al.,1997). To clarify the mechanism by which this effect is achieved, we studied the possible influence of polyamines on the intracellular content of Ca2 + ions - closely involved in the signal transduction pathway.
J Plant PhysioL voL 153. pp. 247-250 (1998)
Materials and Methods
Plant 11IIlteriaJ Protoplasts of sugar beet Uleta vulgaris L.) were isolated byenzymatic digestion of 3-day-old suspensions. They were purified by washing twice in salt solutions (Frearson et al., 1973) without calcium, followed by centrifugation at 75 go, and maintained in modified Murashige and Skoog medium (mMS) (1962) without calcium chloride. Protoplasts purified by washing in calcium-containing salts and maintained in mMS with 3 mmollL Cach were also obtained as a control sample.
Measurements ofCTC-Ca fluorescence Microspectrofluorometric measurements of CTC-Ca fluorescence were made with a Magical system Ooyce Loebl, England) equipped with a Nikon Diaphot inverted microscope coupled to a CCD camera (Extended ISIS-M). An excitation wavelength of 380 nm was provided by a high pressure 100W mercury lamp and DF13 excitation filter, with a 51OWB40 barrier filter (Glen Spectra Limited,
248
ANNA MAJEWSICA-SAWKA, RAFAI. BUTOWT, and A:u!IcsANDRA NnCl.AS
--_...._----_.
.
----------------~~
II I
i
!.
I
..I - --- - -
.------~----~-- -- - - --- ------- -- - -- ----- --- -- -
-
I
i
I
,. ! I
I
i
I
•
Fap.I-6: ere-Ca fluorescence spectra of protoplast suspensions in media without calcium. The curve between points 0.0 and C shows autofluorescence level, C indicates the moment of erc addition, and P moment of polyamine addition. A conspicuous increase in fluores-
cence occurs in the presence of 50 J.LM of Spm (1), 100 J.LM of Spm (2), 100 ~M Spd (3), whereas 100 ~M of Put causes much smaller changes (4). Control measurements of fluorescence after the addition of erc and Spm to media without protoplasts (5) and previous EGTA treatment of protoplast suspensions (6) show no changes in fluorescence.
Fap. 7-10: Fluorescence of protoplasts in media without calcium after the addition of erc only (7), CTC and Put (8), CTC and Spm (9), or EGTA. CTC and Spm (10).
Polyamines Release Membrane-bound Calcium England). Fluorescence was measured from all cells with 14 X 14 11m apenure using an 8 x ocular and a 10/0.75 Fluarx objective. The intensity of erc-Ca fluorescence was expressed in arbitrary values on a scale of 0 to 100. . Protoflasts were placed on a thin glass holder at a density 1.5 x 10 mL -I, and autofluorescence intensity was measured during 10 s. Then they were loaded with a freshly prepared aqueous solution of chlorotetracycline (10- 4 mollL final concentration) by incubation for 40 s in the dark. The level of erc-Ca fluorescence in protoplasts was again measured for 70s (spectra C). In the next step individual polyamines were added at three concentrations: 50, 100 and 300 IlmollL. Immediately thereafrer, the intensity of erC-Ca fluorescence was monitored for 80 s (spectra P). Control experiments were performed by: a) measuring fluorescence intensity after the addition of erc and polyamine to the medium without protoplasts, b) pretreatment of protoplasts with 5 mmollL EGTA for 5 min before erc and PA were added, and c) addition of 0.2% nonionic detergent Triton X-100 after the protoplasts were loaded with
crc.
Results
The autofluorescence of freshly isolated protoplasts incubated in media without calcium was very weak, below 10 arbitrary units (Figs. 1-6). After the addition of erc it increased more than 2-fold (Figs. 1-4, 7). Exogenous Spd and Spm at concentration of 50 and 100 J.1mollL caused a further significant increase in fluorescence intensity to about 30- 50 units (Figs. 1-3, 9). The addition of 300 J.1mollL of both polyamines resulted in relatively greater fluorescence (not shown). The addition of Put was less effective, producing only a minimal increase in erc fluorescence at all concentrations tested. (Figs. 4, 8). Control measurements of fluorescence after the addition of erc and Spm to the medium without protoplasts showed no changes in fluorescence intensity (Fig. 5). Protoplast pretreatment with EGTA caused no increase in fluorescence intensity after the addition of erc and polyamines (Figs. 6, 10). Triton X-100 initiated a decrease in fluorescence observed after CTC addition. The presence of 3 mmollL calcium in the incubation media caused a more visible increase in fluorescence - to about 70 units - in comparison with Ca2 + -free media (data not shown).
Discussion
The mechanism by which erc responds to Ca2 + is different from that of other Ca2+ indicators (Dixon et al., 1984; Nagasaki and Kasai, 1983). Biological membranes are permeable to the zwitterionic form of CTC, which forms a complex with Ca2+ at a stoichiometric ratio 1: 1. The erC-based method is commonly used to detect submillimolar amounts of calcium ions, i.e. higher concentrations than are present in the cytosol. As pointed out by Tsien (1989), erc forms a complex with Ca2 + inside compartments with a high free ion content in the vicinity of hydrophobic sites such as the membranes. The erC-Ca complex then diffuses to the membranes and starts to fluorescence. The fluorescence intensity of CTC-Ca is proportional to the total erc concentration, to the amount of phospholipid membrane, and to Ca2 + con-
249
tent (Dixon et al., 1984; Millman et al., 1980). In our experiments we always used the same erc concentration and a comparable quantity of membrane, as estimated by the number of protoplasts in each sample. In our study, specificity of CTC fluorescence for the CTC-Ca complex was confirmed by using a EGTA - Ca2 + specific chelator (Tirlapur and Willemse, 1992). Moreover, extraction of membranes with the nonionic detergent Triton X-100 completely abolished erc fluorescence, indicating that it originated from the membranous system of protoplasts (Wolniak et al., 1980). Our observations suggest that spermine and spermidine cause rapid changes in intracellular distribution of Ca2+ whereas putrescine does not function so effectivelly - probably because of the presence of only two positively charged amino groups. Spermine and spermidine may therefore function by displacing calcium ions from calcium-binding sites, such as membrane phospholipids, and in consequence may lead to a transient increase in free Ca2 + content. These findings correlate to our previous physiological data, according to which the polyamines stimulate divisions of protoplastderived cells in sugar beet (Majewska-Sawka et al., 1997). Transient changes or local gradients in ci+ are known to contribute to different mitotic events. Elevated level of calcium promotes metaphase progression, regulates assemblydisassembly of spindle microtubules and stimulates the formation of phragmoplast (Hepler, 1992, 1994). The competitive nature of Ca2 + and polyamines in membrane binding was also suggested by Kaur-Shawney and Galston (1991). Similarly, calcium displacement and effiux from cell walls in Glycine max was reponed to occur after the addition of polyamine to suspension cultures (Young and Kauss, 1983). Relatively greater CTC fluorescence was noted in protoplasts kept in calcium-containing media. This may be due to either enhanced polyamine transport occurring in the presence of calcium (Antognoni et al., 1994), or to Ca2 + influx into the protoplasts and its interference with intracellular calcium stores. It seems reasonable to conclude that calcium ions may be involved in the mechanism of polyamine action in plant cells, as occurs in other growth regulators such as auxins and gibbereHins (Felle, 1988; Bush, 1995; Bush, 1996). Future research will concentrate on the effect of polyamines on the cytosolic content of calcium ions. Acknowledgements
The authors thank Dr. E. Wyroba from Nencki Institute in Warsaw from providing a Magical system, and K. Shashok for correcting the English version of the manuscript. This study was supponed by the Polish Committee for Scientific Research (project no. G P04C08712).
References ANTOGNONI, E, P. CAsALI, R. PISTOCCHI, and N. BAGNI: Amino Acids 6, 301-309 (1994). ANTOGNONI, E, R. PISTOCCHI, P. CAsAu, and N. BAGNI: Plant Physiol. Biochem. 33, 701-708 (1995).
250
ANNA MAJBWSKA-SAWKA, RAPAl. BUTOWT, and AuKSANDRA NnG.AS
BUSH, D. S.: Annu. Rev. Plant Physiol. Plant Mol. BioI. 46, 95-122
MAJEWSKA-SAWKA, A., A. NnG.AS, and E. JAZDZEWSKA: BioI. Plant.
- Planta 199, 89-99 (1996). DIXON, D., N. BRANDT, and D. H. HAYNES: J. BioI. Chern. 259,
MILLMAN, M. S., A. H. CASWELL, and D. H. HAYNES: Mernbr. Biochern. 3, 291-315 (1980). MURASHIGE, T. and E SKOOG: Physiol. Plant. 15,473-497 (1962). NAGASAKI, K. and M. KAsAl: J. Biochern. 94, 1101-1109 (1983). TIBURCIO, A. E, J. L. CAMpos, X. FIGUERAS, and R T. BESFORD: Plant Growth Regul. 12, 331-340 (1993). TIRLAPUR, u. K. and M. T. M. WILLEMSE: Sex. Plant Reprod. 5,
(1995).
13737-13741 H984).
EILERS, R J., J. G. SULUVAN, and R M. SKIRVIN: Plant Cell Rep. 7, 216-219 (1988).
FBLLE, H.: Planta 174,495-499 (1988). FRl!ARSON, E. M., J. B. POWER, and E. C. CocKING: Dev. BioI. 33, 130-137 (1973).
HEPLER, P. K.: Inter. Rev. Plant Cytol. 138,239-268 (1992). - Cell Calcium 16, 322-330 (1994). KAuR-SAWHNEY, R, H. E. FWRES, and A. W. GALSTON: Plant Physiol. 65,368-371 (1980). KAUR-SAWHNEY, R and A. W. GALSTON: In: SLOCUM, R D. and H. E. FWRES (005.): Biochemistry and Physiology of Polyamines in Plants, pp. 201-211. Boca Raton, CRC Press (1991).
39,561-567 (1997).
214-223 (1992).
TSIEN, R Y.: Methods Cell BioI. 30, 127-156 (1989). WOLNIAK, S. M., P. K. HEPLER, and W. T. JACKSON: J. Cell BioI. 87, 23-32 (1980).
YOUNG, D. H. and H. KAuSS: Plant Physiol. 73, 698-702 (1983).
• J••••AL.F.
Short Communication
© 1998 by Gustav Fischer Verlag. Jena
Do Polyamines Release Membrane-bound Calcium in Sugar Beet Protoplasts? ANNA MAJEWS~-SAWKA,
RAFAI. BUTOWT, and ALEKSANDRA NU(LAs*
Institute for Plant Breeding and Acclimatization, Powstaflc6w Wielkopolskich 10, 85-950 Bydgoszcz, Poland
* Permanent address: University of Technology and Agriculture, Kaliskiego 7, 85-791 Bydgoszcz, Poland Received March 17, 1997 . Accepted August 26, 1997
Summary The possible effect of exogenous polyamines on the changes in calcium ion content in sugar beet protoplasts maintained in calcium-free media was studied. We combined the chlorotetracycline fluorescent method (CTC) with direct measurements of Ca2+ levels in a Magical system. St<=rmine and spermidine at concentrations of 50, ,100 and 300 J.UI1ollL caused a conspicuous increase of Ca + detectable in the vicinity of membranes, whereas putrescine had no such effect. The specificity of fluorescence for CTC-Ca complex was confirmed by EGTA treatment, and the membranous origin of fluorescence was confirmed by the fact that it was abolished by Triton X-100. Polyamines are therefore postulated to act by displacing Ca2 + from intracellular membranes.
Key words: Beta vulgaris L., calcium, polyamines, protoplasts. Abbreviations: CTC = chlorotetracycline; EGTA = ethylene glycol-bis-(-aminoethylether)- N,N,N',N'tetraacetic acid; mMS = modified Murashige and Skoog medium; Put = putrescine; Spd = spermidine; Spm spermine.
=
Introduction Polyamines affect a wide range of physiological functions in plant cells, including synthesis of RNA, DNA and proteins, stabilization of plasma membranes and transduction of extracellular signals (liburcio et al., 1993). The cellular mechanisms by which these compounds act have been studied extensively in many systems in vitro, and protoplasts are an especially attractive model for experiments with exogenous polyamines (Antognoni et al., 1995). Exogenous spermine and spermidine are rapidly ttanspotted into the protoplasts through a carrier-mediated mechanism (Antognoni et al., 1994), and may stimulate the protoplast division (KaurSawhney et al., 1980; Eilers et al., 1988; Majewska-Sawka et al.,1997). To clarify the mechanism by which this effect is achieved, we studied the possible influence of polyamines on the intracellular content of Ca2 + ions - closely involved in the signal transduction pathway.
J Plant PhysioL voL 153. pp. 247-250 (1998)
Materials and Methods
Plant 11IIlteriaJ Protoplasts of sugar beet Uleta vulgaris L.) were isolated byenzymatic digestion of 3-day-old suspensions. They were purified by washing twice in salt solutions (Frearson et al., 1973) without calcium, followed by centrifugation at 75 go, and maintained in modified Murashige and Skoog medium (mMS) (1962) without calcium chloride. Protoplasts purified by washing in calcium-containing salts and maintained in mMS with 3 mmollL Cach were also obtained as a control sample.
Measurements ofCTC-Ca fluorescence Microspectrofluorometric measurements of CTC-Ca fluorescence were made with a Magical system Ooyce Loebl, England) equipped with a Nikon Diaphot inverted microscope coupled to a CCD camera (Extended ISIS-M). An excitation wavelength of 380 nm was provided by a high pressure 100W mercury lamp and DF13 excitation filter, with a 51OWB40 barrier filter (Glen Spectra Limited,
248
ANNA MAJEWSICA-SAWKA, RAFAI. BUTOWT, and A:u!IcsANDRA NnCl.AS
--_...._----_.
.
----------------~~
II I
i
!.
I
..I - --- - -
.------~----~-- -- - - --- ------- -- - -- ----- --- -- -
-
I
i
I
,. ! I
I
i
I
•
Fap.I-6: ere-Ca fluorescence spectra of protoplast suspensions in media without calcium. The curve between points 0.0 and C shows autofluorescence level, C indicates the moment of erc addition, and P moment of polyamine addition. A conspicuous increase in fluores-
cence occurs in the presence of 50 J.LM of Spm (1), 100 J.LM of Spm (2), 100 ~M Spd (3), whereas 100 ~M of Put causes much smaller changes (4). Control measurements of fluorescence after the addition of erc and Spm to media without protoplasts (5) and previous EGTA treatment of protoplast suspensions (6) show no changes in fluorescence.
Fap. 7-10: Fluorescence of protoplasts in media without calcium after the addition of erc only (7), CTC and Put (8), CTC and Spm (9), or EGTA. CTC and Spm (10).
Polyamines Release Membrane-bound Calcium England). Fluorescence was measured from all cells with 14 X 14 11m apenure using an 8 x ocular and a 10/0.75 Fluarx objective. The intensity of erc-Ca fluorescence was expressed in arbitrary values on a scale of 0 to 100. . Protoflasts were placed on a thin glass holder at a density 1.5 x 10 mL -I, and autofluorescence intensity was measured during 10 s. Then they were loaded with a freshly prepared aqueous solution of chlorotetracycline (10- 4 mollL final concentration) by incubation for 40 s in the dark. The level of erc-Ca fluorescence in protoplasts was again measured for 70s (spectra C). In the next step individual polyamines were added at three concentrations: 50, 100 and 300 IlmollL. Immediately thereafrer, the intensity of erC-Ca fluorescence was monitored for 80 s (spectra P). Control experiments were performed by: a) measuring fluorescence intensity after the addition of erc and polyamine to the medium without protoplasts, b) pretreatment of protoplasts with 5 mmollL EGTA for 5 min before erc and PA were added, and c) addition of 0.2% nonionic detergent Triton X-100 after the protoplasts were loaded with
crc.
Results
The autofluorescence of freshly isolated protoplasts incubated in media without calcium was very weak, below 10 arbitrary units (Figs. 1-6). After the addition of erc it increased more than 2-fold (Figs. 1-4, 7). Exogenous Spd and Spm at concentration of 50 and 100 J.1mollL caused a further significant increase in fluorescence intensity to about 30- 50 units (Figs. 1-3, 9). The addition of 300 J.1mollL of both polyamines resulted in relatively greater fluorescence (not shown). The addition of Put was less effective, producing only a minimal increase in erc fluorescence at all concentrations tested. (Figs. 4, 8). Control measurements of fluorescence after the addition of erc and Spm to the medium without protoplasts showed no changes in fluorescence intensity (Fig. 5). Protoplast pretreatment with EGTA caused no increase in fluorescence intensity after the addition of erc and polyamines (Figs. 6, 10). Triton X-100 initiated a decrease in fluorescence observed after CTC addition. The presence of 3 mmollL calcium in the incubation media caused a more visible increase in fluorescence - to about 70 units - in comparison with Ca2 + -free media (data not shown).
Discussion
The mechanism by which erc responds to Ca2 + is different from that of other Ca2+ indicators (Dixon et al., 1984; Nagasaki and Kasai, 1983). Biological membranes are permeable to the zwitterionic form of CTC, which forms a complex with Ca2+ at a stoichiometric ratio 1: 1. The erC-based method is commonly used to detect submillimolar amounts of calcium ions, i.e. higher concentrations than are present in the cytosol. As pointed out by Tsien (1989), erc forms a complex with Ca2 + inside compartments with a high free ion content in the vicinity of hydrophobic sites such as the membranes. The erC-Ca complex then diffuses to the membranes and starts to fluorescence. The fluorescence intensity of CTC-Ca is proportional to the total erc concentration, to the amount of phospholipid membrane, and to Ca2 + con-
249
tent (Dixon et al., 1984; Millman et al., 1980). In our experiments we always used the same erc concentration and a comparable quantity of membrane, as estimated by the number of protoplasts in each sample. In our study, specificity of CTC fluorescence for the CTC-Ca complex was confirmed by using a EGTA - Ca2 + specific chelator (Tirlapur and Willemse, 1992). Moreover, extraction of membranes with the nonionic detergent Triton X-100 completely abolished erc fluorescence, indicating that it originated from the membranous system of protoplasts (Wolniak et al., 1980). Our observations suggest that spermine and spermidine cause rapid changes in intracellular distribution of Ca2+ whereas putrescine does not function so effectivelly - probably because of the presence of only two positively charged amino groups. Spermine and spermidine may therefore function by displacing calcium ions from calcium-binding sites, such as membrane phospholipids, and in consequence may lead to a transient increase in free Ca2 + content. These findings correlate to our previous physiological data, according to which the polyamines stimulate divisions of protoplastderived cells in sugar beet (Majewska-Sawka et al., 1997). Transient changes or local gradients in ci+ are known to contribute to different mitotic events. Elevated level of calcium promotes metaphase progression, regulates assemblydisassembly of spindle microtubules and stimulates the formation of phragmoplast (Hepler, 1992, 1994). The competitive nature of Ca2 + and polyamines in membrane binding was also suggested by Kaur-Shawney and Galston (1991). Similarly, calcium displacement and effiux from cell walls in Glycine max was reponed to occur after the addition of polyamine to suspension cultures (Young and Kauss, 1983). Relatively greater CTC fluorescence was noted in protoplasts kept in calcium-containing media. This may be due to either enhanced polyamine transport occurring in the presence of calcium (Antognoni et al., 1994), or to Ca2 + influx into the protoplasts and its interference with intracellular calcium stores. It seems reasonable to conclude that calcium ions may be involved in the mechanism of polyamine action in plant cells, as occurs in other growth regulators such as auxins and gibbereHins (Felle, 1988; Bush, 1995; Bush, 1996). Future research will concentrate on the effect of polyamines on the cytosolic content of calcium ions. Acknowledgements
The authors thank Dr. E. Wyroba from Nencki Institute in Warsaw from providing a Magical system, and K. Shashok for correcting the English version of the manuscript. This study was supponed by the Polish Committee for Scientific Research (project no. G P04C08712).
References ANTOGNONI, E, P. CAsALI, R. PISTOCCHI, and N. BAGNI: Amino Acids 6, 301-309 (1994). ANTOGNONI, E, R. PISTOCCHI, P. CAsAu, and N. BAGNI: Plant Physiol. Biochem. 33, 701-708 (1995).
250
ANNA MAJBWSKA-SAWKA, RAPAl. BUTOWT, and AuKSANDRA NnG.AS
BUSH, D. S.: Annu. Rev. Plant Physiol. Plant Mol. BioI. 46, 95-122
MAJEWSKA-SAWKA, A., A. NnG.AS, and E. JAZDZEWSKA: BioI. Plant.
- Planta 199, 89-99 (1996). DIXON, D., N. BRANDT, and D. H. HAYNES: J. BioI. Chern. 259,
MILLMAN, M. S., A. H. CASWELL, and D. H. HAYNES: Mernbr. Biochern. 3, 291-315 (1980). MURASHIGE, T. and E SKOOG: Physiol. Plant. 15,473-497 (1962). NAGASAKI, K. and M. KAsAl: J. Biochern. 94, 1101-1109 (1983). TIBURCIO, A. E, J. L. CAMpos, X. FIGUERAS, and R T. BESFORD: Plant Growth Regul. 12, 331-340 (1993). TIRLAPUR, u. K. and M. T. M. WILLEMSE: Sex. Plant Reprod. 5,
(1995).
13737-13741 H984).
EILERS, R J., J. G. SULUVAN, and R M. SKIRVIN: Plant Cell Rep. 7, 216-219 (1988).
FBLLE, H.: Planta 174,495-499 (1988). FRl!ARSON, E. M., J. B. POWER, and E. C. CocKING: Dev. BioI. 33, 130-137 (1973).
HEPLER, P. K.: Inter. Rev. Plant Cytol. 138,239-268 (1992). - Cell Calcium 16, 322-330 (1994). KAuR-SAWHNEY, R, H. E. FWRES, and A. W. GALSTON: Plant Physiol. 65,368-371 (1980). KAUR-SAWHNEY, R and A. W. GALSTON: In: SLOCUM, R D. and H. E. FWRES (005.): Biochemistry and Physiology of Polyamines in Plants, pp. 201-211. Boca Raton, CRC Press (1991).
39,561-567 (1997).
214-223 (1992).
TSIEN, R Y.: Methods Cell BioI. 30, 127-156 (1989). WOLNIAK, S. M., P. K. HEPLER, and W. T. JACKSON: J. Cell BioI. 87, 23-32 (1980).
YOUNG, D. H. and H. KAuSS: Plant Physiol. 73, 698-702 (1983).