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Red light-mediated changes in inositol phospholipids and phosphatidylcholine in Brassica hypocotyls
Phytochemisrry, Vol. 32, No. 4, pp. 823 -825, 1993 Printedm Great Britain.
003 l-9422:93 $6.00 + 0.00 (0 1993 PergamonPress Ltd
RED LIGHT-MEDIATED CHANGES IN INOSITOL PHOSPHOLIPIDS PHOSPHATIDYLCHOLINE IN BRASSICA HYPOCOTYLS SUMITA PAL, MANOJ KUMAR
ACHARYA
and
AND
SIPRA GUHA-MUKHERJEE*
Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India (Received in revisedform 30 July 1992)
Key Word Index--Brassica oleracea; Cruciferae; hypocotyls; red light-mediated phospholipids; phosphatidylcholine.
changes; inositol
Abstract-We have investigated the effect of a red light stimulus on rapid turnover of phosphoinositides and phosphatidylcholine when incubation was carried out in the dark after removal of the stimulus in etiolated Brassica oleracea hypocotyls. At 30 set there was a remarkable decrease in phosphatidylinositol-4,5_bisphosphate as compared to the control. The amounts of inositol-1,4-bisphosphate and inositol-1,4,5_trisphosphate also decreased at 30 set, whereas there was a sharp increase in inositol-4-monophosphate. There was also a decrease in phosphatidylcholine content within 30 sec. The role of phospholipid turnover in cell proliferation by red light is discussed.
INTRODUCTION
The turnover of phosphoinositides (PI) in the signal transduction pathway is known to be essential for the production of second messengers, cell division and differentiation in animal systems [l]. The involvement of phytochrome in the control of differentiation in Brassica oleracea had already been reported [2] by some studies which showed that red light induced cell proliferation, far-red induced differentiation and also red/far-red reversibility. It has been concluded that these red lightinduced proliferations and far-red light-induced differentiations are likely to be the result of alterations to the plasma membrane PI. Some of the most rapid phytochrome-mediated responses appear to be membranerelated [S]. Hendricks and Borthwich [4] reviewed the phytochrome-mediated physiological effects, suggesting the involvement of membranes as the primary mechanism of action and also stated that these responses are expressed in the range of seconds or minutes. Phytochrome in its Pfr form is capable of binding in uiuo to subcellular structures within a few seconds, being fast enough to account for the fastest physiological effects related to membranes. The effect of a brief white light stimulus on the turnover of PI and phosphatidylcholine (PC) in etiolated B. oleracea seedlings has already been reported from this laboratory [S]. The present study was undertaken to examine the response in PI turnover in seconds when the red light stimulus was removed and the incubation carried out in the dark.
*Author to whom correspondence should be addressed.
RESULTS AND DISCUSSION
When the red light stimulus was given for 5 min (found to be saturating) to the hypocotyls and then kept in the dark for 30 set, there was a decrease in phosphatidylinositol-4,5_bisphosphate (PIP,) (29%), phosphatidylinositol-4-phosphate (PIP) (18%) and PI (18%) compared to the control. A sharp decrease in inositol-1,4,5trisphosphate (IP,) (78%) was also noticed during the same period of time (Fig. 1). The decrease in PIP, could be due to rapid activation of phospholipase C. The plasma membrane bound phospholipase C may be a transducing link in cases when external signals affect metabolic events in plant cells [6]. Murthy et al. [7] have also suggested that GA triggers a rapid stimulation within 15-60 sets of PI biosynthesis followed by rapid breakdown probably due to phospholipase A or C stimulation. A similar report stating that auxin may control a rapid PI turnover in plant cells by its action on phospholipase C has also been published [S]. Recent investigations confirmed the presence of phospholipid-specific phospholipase C in higher plants [9]. The product of phospholipase C catalysed PIP, hydrolysis appears to be involved in some other cellular events, although a direct cause and effect relationship has not yet been established. The decrease in IP, by 78% 30 set after treatment, strongly indicated the activation of phosphatases also with a rapid turnover of IP, to inositol-1,4-bisphosphate (IP,) [lo] and finally to inositol-4-phosphate (IP, ) (64% increase over control) (Fig. 1). Billah et al. [I 1] mentioned in their review that some of the specific phosphatases are activated during agonist stimulation. It has been ascertained by Raymond et al. [12], that phytochrome and blue light receptors change the phosphorylation 823
s. PAL et ai.
824
Table 1. Amounts of labelled IP,, IP, and IP, in 5 mm red light-treated hypocotyls after varying intervals of dark incubation
_. ___ .
% 32 P incorporated ___ _-.. __. _-...-.
(set)
IP,
IPI
IP,
IP, +1P,
0 5 10 20 30 40 50 60
100 123k6.0 136k5.0 153-6.6 164k6.5 138k6.3 96k5.5 ND
100 93+7.7 104i5.0 93+3.1 81k6.2 103k4.3 128k4.0 13Ok5.5
100 9Ok6.5 68k3.6 45+2.6 22k2.5 46,3.5 91It3.0 69+4.7
100 92 89 70 56 76 113 105
Dark incubation
ND
PIP*
After
PIP
30 set
PI
IP2 IP,
_-. -
not determmed.
Table 2. Amounts of labelled PI, PC and PE in 5 min red light-treated hypocotyls following varying interval of dark incubation
of red light treatment
PIP Fig. 1. Amounts of 32P labelled PI (19244mm*), (42.04mrn2), PIP, (16.51 mm’), IP, (1513 cpm), IP, (7972 cpm) IP, (17908 cpm) in 5 min red light-treated hypocotyls after $0 set of dark incubation. 3zP-containing phospholipids (organic phase) were detected by autoradiography and the distribution of these lipids determined by densitometry. Inositol phosphates (aq. phase) were counted for radioactivity.
status of proteins and that phosphorylation/dephosphorylation events are also important in the process of transducing signals to the targeted process within the cell. The coupling of the signal transduction to the second messenger such as phospholipids can in turn regulate the phosphorylation or dephosphorylation events. Time kinetics demonstrated that during dark incubation following by 5 min red light treatment there is a gradual increase in IP, up to 30 set (Table 1). The increase in IP, was less than that of IP,, and there was a sharp decrease in IP, at 30 sec. The same trend is also seen with IP, +IP,. Between 30 and 50 sets, an increase in IP,, IP, and IP2 + IP, were observed with a simultaneous decrease in IP,. It has been shown earlier by us [SJ that after light irradiation of 50 set there is a 40% increase in IP, + IP, compared with the dark control. It is quite possible that the IP,-ase or the phosphatase that converts IP, to IP,, being an integral protein in the plasma membrane, also gets activated rapidly by red light treatment. There was a sharp decrease in PI (30%) within the first 5 set but little change occurred until 50 set (Table 2). The breakdown of PI may be due to the stimulation of phospholipase A (to yield lysophosphatidylinositol) or a phosphohpase C-like phosphodiestrase (to yield inositol-l-phosphate and diacylglycerol) or PI kinase (to yield PIP). In the present investigation there was a significant increase in PC in the first 10 set followed by a rapid decrease (Table 2). Decreases in PC produced by a white light stimulus has been already reported from this laboratory [SJ. Studies on
Dark incubation
_
(sccJ
PI
PC
PE
0 5 10 20 30 40 50 60
100 70 + 4.7 19 + 6.0 ND 8255.1 ND 78 + 5.1 91+7.5
100 11646.5 12Ok5.9 9925.0 78 f 4.0 84+2.5 93 + 6.0 9823.1
100 101 k5.0 104k3.6 ND 109k4.5 ND 107k6.1 10455.5
ND
%32P incorporated into phospholipids _. _ ._ __ ._ _. ._ ._ -. __
not determined.
animal cells [i 11 suggested that in response to an agonist, diacylglycerol (DAG) is produced in a biphasic manner. The delayed or long-lasting phase has been shown to be associated with PC breakdown. Phospholipase C that utilises PC as substrate, has been partially purified from dog heart cytosol [13]. There was not much change in phosphatidylethanolamine (PE) (Table 2) until 60 set after the red light treatment compared to the control. PE, the second most abundant phospholipid in mammalian tissue is poorly degraded by partially purified phospholipase C and D [ 13, 141. In the case of Brassica seedlings a short pulse of white light did not change the PE content by very much [S]. Our investigation supports our earlier data [2,15] that red light stimulates change in PI. However, it does show that changes occur in seconds and thus confirms that it is an early step in the sequence of events triggering biochemical changes leading to cell proliferation.
EXPERIMENTAL
Plant material and growth conditions. Seedlings of B. oleracea var. botrytis cv Synthetic were grown on germination paper in the dark at 25 + 1’ for 7 days.
Red light-mediated changes in phospholipids 32P incorporation and extraction of lipids. Hypocotyls (500 mg) were excised and labelled with 15 $i of orthophosphate for 2 hr. Dark-grown hypocotyls were exposed to red light (intensity 1.5 Wm-‘) for 5 min and then incubated in the dark for different times; control hypocotyls were kept in the dark without red light treatment. Immediately after incubation for varying intervals of time, hypocotyls were rapidly frozen in liquid N, ground in a mortar and pestle and extracted with CHCl,MeOH-3.4 M HCl (6: 6: 1) to separate organic and aq. phases as described in ref. [16]. Separation and identijication of phospholipids. Phospholipids from the organic phase and inositol phosphates from the aq. phase were sepd and identified as described in ref. [S]. Data presentation. Results are mean values from 3 expts. The average of the dark controls was designated as 100%; values for various treatments are expressed as a percentage of the dark control. Acknowledgements-We are grateful for the financial support from the Department of Biotechnology and to CSIR for awarding fellowships to S.P. and M.K.A.. We also appreciate help and useful suggestions from Prof. R. Prasad (JNU).
825
2. Basu, A., Sethi, U. and Guha-Mukherjee,
I. and Guha5. Acharya, M. K., Dureja-Munjal, Mukherjee, S. (1991) Phytochemistry 30, 2895. 6. Melin, P. M., Sommarin, M., Sandelius, A. S. and Jergil, B. (1987) FEBS Letters 223, 87. 7. Murthy, P. P. N., Renders, J. M. and Keranen, L. M. (1989) Plant Physiol. 91, 1266. 8. Zebell, B. and Walter-Back, C. (1988) Plant Physiol. 133, 353.
9. Einspahr, K. J. and Thompson,
G. A. (1990) Plant
Physiol. 93, 361.
10. Memon, A. R., Rincon, M. and Boss, W. F. (1989) Plant Physiol. 91, 477.
11. Billah, M. M. and Anthes, J. C. (1990) Biochem. J. 269, 281. 12. Raymond, J. A. B. and Douglas, D. R. (1990) Plant Physiol. 94, 1501.
13. Wolf, R. A. and Gross, R. W. (1985) J. Biol. Chem. 260, 7295.
14. Taki, T. and Kanfer, J. N. (1979) J. Biol. Chem. 254, 9701.
15. Sethi, U., Basu, A. and Guha-Mukherjee, UEFERENCES
1. Whitman,
M. and Cantley, L. (1988) Biochim. Bio-
phys. Acta. 948, 327.
S. (1990)
Phytochemistry 29, 1539. 3. Marme, D. (1977) Ann. Rev. Plant Physiol. 28, 173. 4. Hendricks, S. B., Borthwick, H. A. (1967) Proc. Nat1 Acad. Sci. USA 58, 2125.
S. (1990)
Phytochemistry 29, 825.
16. Morse, M. J., Crain, R. C. and Satter, R. L. (1987) Proc. Nat1 Acad. Sci. USA. 84, 7075.
003 l-9422:93 $6.00 + 0.00 (0 1993 PergamonPress Ltd
RED LIGHT-MEDIATED CHANGES IN INOSITOL PHOSPHOLIPIDS PHOSPHATIDYLCHOLINE IN BRASSICA HYPOCOTYLS SUMITA PAL, MANOJ KUMAR
ACHARYA
and
AND
SIPRA GUHA-MUKHERJEE*
Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India (Received in revisedform 30 July 1992)
Key Word Index--Brassica oleracea; Cruciferae; hypocotyls; red light-mediated phospholipids; phosphatidylcholine.
changes; inositol
Abstract-We have investigated the effect of a red light stimulus on rapid turnover of phosphoinositides and phosphatidylcholine when incubation was carried out in the dark after removal of the stimulus in etiolated Brassica oleracea hypocotyls. At 30 set there was a remarkable decrease in phosphatidylinositol-4,5_bisphosphate as compared to the control. The amounts of inositol-1,4-bisphosphate and inositol-1,4,5_trisphosphate also decreased at 30 set, whereas there was a sharp increase in inositol-4-monophosphate. There was also a decrease in phosphatidylcholine content within 30 sec. The role of phospholipid turnover in cell proliferation by red light is discussed.
INTRODUCTION
The turnover of phosphoinositides (PI) in the signal transduction pathway is known to be essential for the production of second messengers, cell division and differentiation in animal systems [l]. The involvement of phytochrome in the control of differentiation in Brassica oleracea had already been reported [2] by some studies which showed that red light induced cell proliferation, far-red induced differentiation and also red/far-red reversibility. It has been concluded that these red lightinduced proliferations and far-red light-induced differentiations are likely to be the result of alterations to the plasma membrane PI. Some of the most rapid phytochrome-mediated responses appear to be membranerelated [S]. Hendricks and Borthwich [4] reviewed the phytochrome-mediated physiological effects, suggesting the involvement of membranes as the primary mechanism of action and also stated that these responses are expressed in the range of seconds or minutes. Phytochrome in its Pfr form is capable of binding in uiuo to subcellular structures within a few seconds, being fast enough to account for the fastest physiological effects related to membranes. The effect of a brief white light stimulus on the turnover of PI and phosphatidylcholine (PC) in etiolated B. oleracea seedlings has already been reported from this laboratory [S]. The present study was undertaken to examine the response in PI turnover in seconds when the red light stimulus was removed and the incubation carried out in the dark.
*Author to whom correspondence should be addressed.
RESULTS AND DISCUSSION
When the red light stimulus was given for 5 min (found to be saturating) to the hypocotyls and then kept in the dark for 30 set, there was a decrease in phosphatidylinositol-4,5_bisphosphate (PIP,) (29%), phosphatidylinositol-4-phosphate (PIP) (18%) and PI (18%) compared to the control. A sharp decrease in inositol-1,4,5trisphosphate (IP,) (78%) was also noticed during the same period of time (Fig. 1). The decrease in PIP, could be due to rapid activation of phospholipase C. The plasma membrane bound phospholipase C may be a transducing link in cases when external signals affect metabolic events in plant cells [6]. Murthy et al. [7] have also suggested that GA triggers a rapid stimulation within 15-60 sets of PI biosynthesis followed by rapid breakdown probably due to phospholipase A or C stimulation. A similar report stating that auxin may control a rapid PI turnover in plant cells by its action on phospholipase C has also been published [S]. Recent investigations confirmed the presence of phospholipid-specific phospholipase C in higher plants [9]. The product of phospholipase C catalysed PIP, hydrolysis appears to be involved in some other cellular events, although a direct cause and effect relationship has not yet been established. The decrease in IP, by 78% 30 set after treatment, strongly indicated the activation of phosphatases also with a rapid turnover of IP, to inositol-1,4-bisphosphate (IP,) [lo] and finally to inositol-4-phosphate (IP, ) (64% increase over control) (Fig. 1). Billah et al. [I 1] mentioned in their review that some of the specific phosphatases are activated during agonist stimulation. It has been ascertained by Raymond et al. [12], that phytochrome and blue light receptors change the phosphorylation 823
s. PAL et ai.
824
Table 1. Amounts of labelled IP,, IP, and IP, in 5 mm red light-treated hypocotyls after varying intervals of dark incubation
_. ___ .
% 32 P incorporated ___ _-.. __. _-...-.
(set)
IP,
IPI
IP,
IP, +1P,
0 5 10 20 30 40 50 60
100 123k6.0 136k5.0 153-6.6 164k6.5 138k6.3 96k5.5 ND
100 93+7.7 104i5.0 93+3.1 81k6.2 103k4.3 128k4.0 13Ok5.5
100 9Ok6.5 68k3.6 45+2.6 22k2.5 46,3.5 91It3.0 69+4.7
100 92 89 70 56 76 113 105
Dark incubation
ND
PIP*
After
PIP
30 set
PI
IP2 IP,
_-. -
not determmed.
Table 2. Amounts of labelled PI, PC and PE in 5 min red light-treated hypocotyls following varying interval of dark incubation
of red light treatment
PIP Fig. 1. Amounts of 32P labelled PI (19244mm*), (42.04mrn2), PIP, (16.51 mm’), IP, (1513 cpm), IP, (7972 cpm) IP, (17908 cpm) in 5 min red light-treated hypocotyls after $0 set of dark incubation. 3zP-containing phospholipids (organic phase) were detected by autoradiography and the distribution of these lipids determined by densitometry. Inositol phosphates (aq. phase) were counted for radioactivity.
status of proteins and that phosphorylation/dephosphorylation events are also important in the process of transducing signals to the targeted process within the cell. The coupling of the signal transduction to the second messenger such as phospholipids can in turn regulate the phosphorylation or dephosphorylation events. Time kinetics demonstrated that during dark incubation following by 5 min red light treatment there is a gradual increase in IP, up to 30 set (Table 1). The increase in IP, was less than that of IP,, and there was a sharp decrease in IP, at 30 sec. The same trend is also seen with IP, +IP,. Between 30 and 50 sets, an increase in IP,, IP, and IP2 + IP, were observed with a simultaneous decrease in IP,. It has been shown earlier by us [SJ that after light irradiation of 50 set there is a 40% increase in IP, + IP, compared with the dark control. It is quite possible that the IP,-ase or the phosphatase that converts IP, to IP,, being an integral protein in the plasma membrane, also gets activated rapidly by red light treatment. There was a sharp decrease in PI (30%) within the first 5 set but little change occurred until 50 set (Table 2). The breakdown of PI may be due to the stimulation of phospholipase A (to yield lysophosphatidylinositol) or a phosphohpase C-like phosphodiestrase (to yield inositol-l-phosphate and diacylglycerol) or PI kinase (to yield PIP). In the present investigation there was a significant increase in PC in the first 10 set followed by a rapid decrease (Table 2). Decreases in PC produced by a white light stimulus has been already reported from this laboratory [SJ. Studies on
Dark incubation
_
(sccJ
PI
PC
PE
0 5 10 20 30 40 50 60
100 70 + 4.7 19 + 6.0 ND 8255.1 ND 78 + 5.1 91+7.5
100 11646.5 12Ok5.9 9925.0 78 f 4.0 84+2.5 93 + 6.0 9823.1
100 101 k5.0 104k3.6 ND 109k4.5 ND 107k6.1 10455.5
ND
%32P incorporated into phospholipids _. _ ._ __ ._ _. ._ ._ -. __
not determined.
animal cells [i 11 suggested that in response to an agonist, diacylglycerol (DAG) is produced in a biphasic manner. The delayed or long-lasting phase has been shown to be associated with PC breakdown. Phospholipase C that utilises PC as substrate, has been partially purified from dog heart cytosol [13]. There was not much change in phosphatidylethanolamine (PE) (Table 2) until 60 set after the red light treatment compared to the control. PE, the second most abundant phospholipid in mammalian tissue is poorly degraded by partially purified phospholipase C and D [ 13, 141. In the case of Brassica seedlings a short pulse of white light did not change the PE content by very much [S]. Our investigation supports our earlier data [2,15] that red light stimulates change in PI. However, it does show that changes occur in seconds and thus confirms that it is an early step in the sequence of events triggering biochemical changes leading to cell proliferation.
EXPERIMENTAL
Plant material and growth conditions. Seedlings of B. oleracea var. botrytis cv Synthetic were grown on germination paper in the dark at 25 + 1’ for 7 days.
Red light-mediated changes in phospholipids 32P incorporation and extraction of lipids. Hypocotyls (500 mg) were excised and labelled with 15 $i of orthophosphate for 2 hr. Dark-grown hypocotyls were exposed to red light (intensity 1.5 Wm-‘) for 5 min and then incubated in the dark for different times; control hypocotyls were kept in the dark without red light treatment. Immediately after incubation for varying intervals of time, hypocotyls were rapidly frozen in liquid N, ground in a mortar and pestle and extracted with CHCl,MeOH-3.4 M HCl (6: 6: 1) to separate organic and aq. phases as described in ref. [16]. Separation and identijication of phospholipids. Phospholipids from the organic phase and inositol phosphates from the aq. phase were sepd and identified as described in ref. [S]. Data presentation. Results are mean values from 3 expts. The average of the dark controls was designated as 100%; values for various treatments are expressed as a percentage of the dark control. Acknowledgements-We are grateful for the financial support from the Department of Biotechnology and to CSIR for awarding fellowships to S.P. and M.K.A.. We also appreciate help and useful suggestions from Prof. R. Prasad (JNU).
825
2. Basu, A., Sethi, U. and Guha-Mukherjee,
I. and Guha5. Acharya, M. K., Dureja-Munjal, Mukherjee, S. (1991) Phytochemistry 30, 2895. 6. Melin, P. M., Sommarin, M., Sandelius, A. S. and Jergil, B. (1987) FEBS Letters 223, 87. 7. Murthy, P. P. N., Renders, J. M. and Keranen, L. M. (1989) Plant Physiol. 91, 1266. 8. Zebell, B. and Walter-Back, C. (1988) Plant Physiol. 133, 353.
9. Einspahr, K. J. and Thompson,
G. A. (1990) Plant
Physiol. 93, 361.
10. Memon, A. R., Rincon, M. and Boss, W. F. (1989) Plant Physiol. 91, 477.
11. Billah, M. M. and Anthes, J. C. (1990) Biochem. J. 269, 281. 12. Raymond, J. A. B. and Douglas, D. R. (1990) Plant Physiol. 94, 1501.
13. Wolf, R. A. and Gross, R. W. (1985) J. Biol. Chem. 260, 7295.
14. Taki, T. and Kanfer, J. N. (1979) J. Biol. Chem. 254, 9701.
15. Sethi, U., Basu, A. and Guha-Mukherjee, UEFERENCES
1. Whitman,
M. and Cantley, L. (1988) Biochim. Bio-
phys. Acta. 948, 327.
S. (1990)
Phytochemistry 29, 1539. 3. Marme, D. (1977) Ann. Rev. Plant Physiol. 28, 173. 4. Hendricks, S. B., Borthwick, H. A. (1967) Proc. Nat1 Acad. Sci. USA 58, 2125.
S. (1990)
Phytochemistry 29, 825.
16. Morse, M. J., Crain, R. C. and Satter, R. L. (1987) Proc. Nat1 Acad. Sci. USA. 84, 7075.