- Email: [email protected]
Enzymatic activities of the phosphatidylinositol cycle during growth of suspension cultured plant cells
Plant Science, 49 (1987) 167--173 Elsevier Scientific Publishers Ireland Ltd.
167
ENZYMATIC ACTIVITIES OF THE PHOSPHATIDYLINOSITOL CYCLE DURING GROWTH OF SUSPENSION CULTURED PLANT CELLS
SABINA HELM and KARL G. WAGNER*
GBF, Arbeitgsgruppe Enzymologie, D-3300 Braunschweig (F.R.G.) (Received September 1st, 1986) (Revision received November 13th, 1986) (Accepted November 13th, 1986) Determination of individual phospholipids during the growth of a Catharanthus roseus suspension culture showed a very strong transient increase in the PI and PA content during the cell division phase. This indicates a high turnover of these phospholipids, moat likely promoted via the phosphatidylinositol cycle through phosphorylation of the inoaitol head group. Phospholipid phosphorylation b y exogenous ATP was followed with cells harvested at different points of the growth cycle. The phosphoUpid kinases showed peak activities in the cell division phase, whereas in the cell elongation phase lower hut significant activities were observed. These results document that the phosphatidylinositol cycle is operating in cultured plant cells and obviously plays a role in growth regulation.
Key words: phospholipid kinases; phosphatidylinositol cycle; growth cycle; suspension cultured plant cells; Catharanthus roseus
Introduction Plant cells grow by cell division and cell elongation, growth phases which can be studied with suspension cultured cells [1,2]. Molecular mechanisms of growth regulation are widely unknown for plant cells,whereas in the field of animal cells several, although not completely understood, concepts exist. It has been shown that the so called phosphatidylinositol cycle (PI cycle) is involved in the growth regulation of animal or yeast cells [3,4]. Boss and Massel [5], Strasser et al. [6] and Heim and Wagner [7] have shown that phosphorylated phosphatidylinositols, which initiate the PI cycle (cf. conclusions, Fig. 4), are also present in cultured plant cells. Moreover we have described
*To w h o m correspondence should be sent. Abbreviations: D A G , diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol; PIP and PIP2, the mono- and diphosphorylated forms of PI.
a method that allows measurement in situ of the phospholipid kinases of the PI cycle with exogenous and labeled ATP and endogenous phospholipid substrates [8]. Furthermore the existence of PI kinases has also been shown in isolated membrane fractions from barley hypocotyls [9]. In the present work we determined phospholipid kinase activities in the growth cycle of suspension cultured Catharanthus roseus cells; in addition the PI and P A content was measured during growth. In conclusion these results showed, that PI is turned over very rapidly, the degree of turnover and PI content strongly correlates with the cell division phase of the growth cycle. Materials and methods Origin and growth of suspension cultured cells has been described [10]. The cells were harvested along the growth cycle, quickly filtered, washed with cold water, frozen with liquid nitrogen and stored at--70°C.
0618o9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
168 Phosphorylation with exogenous [7-32P]ATP, extraction of phospholipids, separation by HPTLC and determination of phosphate incorporated into the individual components has been described recently [8].
Analysis of individual phospholipid components Extraction of 1 g frozen cells with 3.8 ml CHC13/CHaOH (1:2) containing 1% concentrated HC1 was performed in a PotterElvehjem homogenizer. After addition of 1.2 ml CHC13 and 1.2 ml 2 M KC1, incubation at ambient temperatures for 1 h, centrifugation, washing of the lower phase with 2 ml CH3OH/10 mM KH2PO4/CHC13 (48 : 47 : 3) containing 0.25% HC1 and evaporation, the residue was dissolved in 100 ~l CHC13 [11]. Separation of the phospholipids was performed with 20 × 20 cm silica gel thin layer plates F 1500 from Schleicher and Schuell according to Ref. 12. After a prerun with CH3OH, spotting of the samples with microcaps, drying in a dessicator filled with silica gel at 4°C for 3 h, the plates were developed with CHC13/CH3OH/glacial acetic acid/0.9% NaC1 ( 5 0 : 2 5 : 8 : 2 . 5 ) at 4°C. Quantitative determination of the individual components was performed according to Ref. 13. The plates were sprayed with a solution of 0.035% 6-p-toluidino-2-naphthalenesulfonic acid in 0.1 M Tris--I-IC1 of pH 7.4, dried and the spots were scanned at Aa~s nm w i t h t h e fluorescence scanner CS-920 from Shimadzu. With each determination several different amounts of reference phospholipids were spotted on the same plate, developed and scanned, in order to establish a suitable calibration. Results and discussion
Phosphate incorporatio n in the total phospholipids As described in previous work [8] suspension cultured cells of C. roseus, after freezing and thawing twice, incorporate 32p from exogenous ATP into their phospholipid fraction due to phospholipid kinases
which obviously act in situ on endogenous lipid substrate. It was further shown [8] that the kinetics of this phosphorylation is rather fast with a short linear phase and leveling off after about 5 rain at 25°C. For the activity determination of these phospho. lipid kinases, in the present work, incubation was performed for 2 rain only to ensure linear time kinetics. In Fig. 1 cells were harvested at different times during the growth cycle and their phospholipid phosphorylation activity was determined. Incorporation into the total phospholipid fraction shows a very interesting profile with a peak activity at day 5. It has been shown [1,2] that the growth cycle of a plant suspension culture can be divided into 4 phases: after the initial lag phase and the subsequent cell division phase a further growth phase follows which is due mostly to cell elongation caused by the enlargement of the vacuole. Whereas the cell division phase is limited by the amount of the medium phosphate, the cell elongation phase is limited by the size of the carbon source [2]. When the accumulated carbon source decreases below a small threshold value, the starvation phase begins [2]. There is an exponential increase in the phospholipid phosphorylation activity (Fig. 1) in the lag and cell division phase. The decrease obviously indicates the transition to the cell elongation phase with a drop to very low activity at starvation.
Incorporation into the individual components Resolution of the phosphorylated phospholipids by thin layer chromatography reveals a very simple picture [81. The main incorporation (Fig. 2A) from ATP occurs by a phosphatidylinositol kinase which leads to monophosphorylated phosphatidylinositol (PIP) and by a diacylglycerol kinase leading to phosphatidic acid (PA). There is less phosphate incorporation into the diphosphorylated phosphatidylinositol (PIP2) and into phosphatidylinositol (PI). The latter corn-
169
A
FW Phosphotipids P-32 from
E
ATP
0 0 I A
"7
0
o 04-
c
E
04 0 cO
0
.c
v
n
II bo o
o_
-6 E
b
0
1 2
I Z.
I 6
I 8
f 10
r 12
~u a " ys
Fig. I. Phosphate incorporation from labeled A T P into the phospholipid fraction of C. roseus cells during the growth cycle. Phosphorylation was performed as described (cf. Materials and methods and Ref. 8), incubation time was 2 min. Growth cycle (,) in g fresh wt. per 80 ml medium.
pound cannot be obtained directly by the action of a kinase, it reflects however PI synthesis from previously labeled PA which occurs in the endoplasmic reticulum [14]. The phosphorylation pattern is very similar to that obtained with animal cells and exogenous ATP [15]. It is interesting that the peak activity for PA labeling occurs about 2 days before the peak activity of PIP labeling, although we cannot give an explanation for this fact at present. The apparent phospholipid phosphorylation activities of Figs. 1 and 2A were related to 100 mg fresh wt. In order to eliminate changes generated by dilution through the increase of cell mass during the growth cycle, phosphorylation activities were related
to the whole growing population (Fig. 2B). The data in Fig. 2B show that the main activities, i.e. PI kinase (PIP) and diacylglycerol kinase (PA), both increase up to day 7 which probably corresponds to the end of the cell division phase. In the subsequent elongation phase the activities, correlated to the whole population, attain lower but almost constant levels and only in the starvation phase do the activities decrease to rather low values. Obviously during cell elongation there is no net synthesis of these phospholipid kinases, although the vacuolar and plasmalemma m e m brane increase strongly in this growth phase. But it is possible that in this growth phase components for new membrane synthesis
170
(,,) ~,-(1~08) OC I
/W_-I 6
oE
~
o
•
Ot I
I
(33
0_" n LL
-\
n n
n
o
~ ~ "~ 0~
o~
I
o~: L-(I~OIB)
o~
u!~/=u!d
lO~U
•~
0
...-k~
o
8 . ~ 0 ' ~
C0
EO
!.0
~_(6uuOOl)~-(u!uJz) °~d IO~U
0~
171
are recruited by degrading other cell membranes.
measured. An evaluation of this question is possible from the data of Fig. 3, which show- the PI content during the growth cycle. Related to the whole cell population about 0.5--2 mg PI are present which corresponds to 0.6--2.3 ~ m o l PI calculated on the basis of a dioleate c o m p o u n d (MW = 863). The data of Fig. 2B show total activities for the PI kinase of up to 40 nmol • min -1. As enzyme kinetics was followed for only 2 rain and PI is present in the # m o l
PI and P A content during the growth cycle In the present work the activity of the phospholipid kinases was determined by exogenous A T P and endogenous lipid substrates. It was confirmed [8] that A T P is not limiting, the concentration of the endogenous lipid substrate, however, could be limiting, although initial rates (2 rain) were
PA
<3 d
4 -30 v
E
© co
E
3-
O co v
-20
PI
Q_ 0
L O
2_
c~
..c o_ u~
0 _c U~
-10 I
E
I
I
I
I
I
I
I
0
2
/.
6
8
10
12
days Fig. 3. Content of PI and P A in the growth cycle of C. roseus cells.The lipidswere separated and determined as described in Materials and methods. Growth cycle (=) in g fresh wt. per 80 ml medium.
172
range endogenous substrate should not be limited in this case. This estimation, however, is only correct with the assumption that the endogenous lipid substrates are freely available to the phospholipid kinases measured in situ. Figure 3 shows that the PI and PA content varies strongly during the growth phase. As these values are related to the whole cell population, changes by dilution during growth are corrected for. Hence the data reveal a strong increase in the absolute content during the lag phase and cell division phase and a decrease in the cell elongation phase. This indicates that only cell division is correlated both with high enzymatic activities (Fig. 2B) and substrate constituents (PI and PA) of the PI cycle, whereas the cell elongation phase has lower but significant enzymatic activities (Fig. 2B) and a decreasing content of PA and PI (Fig. 3). Obviously regulation mechanisms exist which not only control the degree of the enzymatic activities but also the pool sizes of the lipid substrates of the PI cycle, unless both control mechanisms are linked. Conclusions
operating PI cycle is essential for cell division also in the plant kingdom. Figure 4 illustrates the PI cycle with its phospholipid constituents only. The hydrophflic head groups split off by phospholipase C (e.g. inositol-P3 from PIP2) are not shown. There is evidence, however, that these inositol phosphates are present in growing C. roseus cell cultures (S. Helm, unpublished results). The cell elongation phase starts when the cellular phosphate pool decreases below a low threshold value provided the C~ource is not exhausted; it is characterized by a halt in nucleotide, protein and RNA synthesis, and by a halt in net increase of cytoplasmic volume [2,16]. We have also shown that protein kinase activities decrease in this phase [10] and the present work shows that the constituents of the PI cycle (PI and PA) also decrease, whereas the phospholipid kinase activities assume a lower but significant level. Obviously the PI cycle (Fig. 4) is still operating in this phase although with reduced activity and only at starvation is this cycle shut off. The total activities of the phospholipid kinases (Fig. 2B) are rather high. Incorporation of phosphate from ATP into PIP
In previous work on N i c o t i a n a [16] and Datura [2] cell cultures we showed that the cell division phase is characterized by exponential increases in the nucleotide pool, RNA and protein content, which obviously reflects the increases in net cytoplasmic volume. It has been further shown that in Murashige/Skoog [ 17] and Linsmaier/Skoog media [18] the phosphate source, i.e. the cellular phosphate pool sets a limit on the duration of the cell division phase [2,19]. Among the early enzymes synthesized in the cell division phase are protein kinases [10] and as shown with the present work also the phospholipid kinases of the PI cycle. Figure 3 further reveals that the components of this cycle, such as PA and PI also increase exponentially in the cell division phase. This in conclusion indicates that an actively
ATP
II
DAG
"°.. . . . . . . . . . . . . . . . . .
....°°"
Fig. 4. Phosphatidylinositol cycle. This shows only the phospholipid constituents; ponents determined in the present work cated in boxes. DAG diacylglycerol; [C] lipase C.
pathway the comare indiphospho-
173 attains a value o f a b o u t 40 n m o l • min -1, this is a b o u t 2% of t he total PI c o n t e n t which is p h o s p h o r y l a t e d in 1 rain. A similar value m a y be o b t ai ned f or the p h o s p h o r y l ation o f diacylglycerol, although we have no data o f the n e t c o n t e n t of this lipid. However, the high c o n t e n t of PA (Fig. 3) supports the supposition t h a t diacylglycerol is t u r n e d over very fast. In conclusion this data indicate a r ath er high rate for PI cycle at least in the cell division phase. Acknowledgements
We a r e grateful to Dr. V. Wray for linguistic advice, t o Mrs. H. Starke for typing t he m a n u s c r i p t and Mrs. C. Lippelt for preparing the graphs. This w o r k has been s u p p o r t e d b y t h e Deutsche Forschungsgemeninschaft (Wa 9 1 / 1 5 ) and t he Fonds der Chemischen Industrie. References
4 P.W. Majerus, D.B. Wilson, T.M. Connolly, T.E. Bross and E.J. Neufeld, Trends Biochem. Sci., 10 (1985) 168. 5 W.F. Boss and M.O. Massel, Biochem. Biophys. Res. Commun., 132 (1985) 1018. 6 H. Stasser, C. Hoffmann, H. Grisebach and U. Matern, Z. Naturforsch., 41c (1986) 717. 7 S. Helm and K.G. Wagner, Biochem. Biophys. Res. Commun., 134 (1986) 1175. 8 S. Helm, A. Bauleke, C. Wylegalla and K.G. Wagner, Plant Sci.,49 (1987) 159. 9 A.S. SandeUus and M. Sommarin, F E B S Lett.,
201 (1986) 282. 10 S. Helm, H. Erdmann and K.G. Wagner, Plant Sci., 42 (1985) 159. 11 I. Litosch, S.-H. Lin and J.N. Fain, J. Biol. Chem., 258 (1983) 13727. 12 M. Goppelt and K. Resch, Anal. Biochem., 140 (1984) 152. 13 M. Jones, R.W. Keenan and P. Horowitz, J. Chromatogr., 237 (1982) 522, 14 J.B. Mudd, in: P.K. Stumpf and E.E. Conn (Eds.), Biochem. Plants, Vol. 4, Academic Press, N.Y., 1980, p. 249. 15 M.E. Dunlop and R.G. Larkins, J. Biol. Chem., 259 (1984) 8407. 16 R. Meyer and K.G. Wagner, Physiol. Plant., 65 (1985) 439. 17 T. Murashige and F. Skoog, Physiol. Plant., 15
1 H.E. Street, Tissue Culture and Plant Science, Academic Press, London, 1974. 2 C. WylegaUa, R. Meyer and K.G. Wagner, Planta, 166 (1985) 446. 3 M.J. Berridge, Biochem. J., 220 (1984) 345.
(1962) 473. 18 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 19 S. Amino, T. Fujimura and A. Komamine, Physiol. Plant., 59 (1983) 393.
167
ENZYMATIC ACTIVITIES OF THE PHOSPHATIDYLINOSITOL CYCLE DURING GROWTH OF SUSPENSION CULTURED PLANT CELLS
SABINA HELM and KARL G. WAGNER*
GBF, Arbeitgsgruppe Enzymologie, D-3300 Braunschweig (F.R.G.) (Received September 1st, 1986) (Revision received November 13th, 1986) (Accepted November 13th, 1986) Determination of individual phospholipids during the growth of a Catharanthus roseus suspension culture showed a very strong transient increase in the PI and PA content during the cell division phase. This indicates a high turnover of these phospholipids, moat likely promoted via the phosphatidylinositol cycle through phosphorylation of the inoaitol head group. Phospholipid phosphorylation b y exogenous ATP was followed with cells harvested at different points of the growth cycle. The phosphoUpid kinases showed peak activities in the cell division phase, whereas in the cell elongation phase lower hut significant activities were observed. These results document that the phosphatidylinositol cycle is operating in cultured plant cells and obviously plays a role in growth regulation.
Key words: phospholipid kinases; phosphatidylinositol cycle; growth cycle; suspension cultured plant cells; Catharanthus roseus
Introduction Plant cells grow by cell division and cell elongation, growth phases which can be studied with suspension cultured cells [1,2]. Molecular mechanisms of growth regulation are widely unknown for plant cells,whereas in the field of animal cells several, although not completely understood, concepts exist. It has been shown that the so called phosphatidylinositol cycle (PI cycle) is involved in the growth regulation of animal or yeast cells [3,4]. Boss and Massel [5], Strasser et al. [6] and Heim and Wagner [7] have shown that phosphorylated phosphatidylinositols, which initiate the PI cycle (cf. conclusions, Fig. 4), are also present in cultured plant cells. Moreover we have described
*To w h o m correspondence should be sent. Abbreviations: D A G , diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol; PIP and PIP2, the mono- and diphosphorylated forms of PI.
a method that allows measurement in situ of the phospholipid kinases of the PI cycle with exogenous and labeled ATP and endogenous phospholipid substrates [8]. Furthermore the existence of PI kinases has also been shown in isolated membrane fractions from barley hypocotyls [9]. In the present work we determined phospholipid kinase activities in the growth cycle of suspension cultured Catharanthus roseus cells; in addition the PI and P A content was measured during growth. In conclusion these results showed, that PI is turned over very rapidly, the degree of turnover and PI content strongly correlates with the cell division phase of the growth cycle. Materials and methods Origin and growth of suspension cultured cells has been described [10]. The cells were harvested along the growth cycle, quickly filtered, washed with cold water, frozen with liquid nitrogen and stored at--70°C.
0618o9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
168 Phosphorylation with exogenous [7-32P]ATP, extraction of phospholipids, separation by HPTLC and determination of phosphate incorporated into the individual components has been described recently [8].
Analysis of individual phospholipid components Extraction of 1 g frozen cells with 3.8 ml CHC13/CHaOH (1:2) containing 1% concentrated HC1 was performed in a PotterElvehjem homogenizer. After addition of 1.2 ml CHC13 and 1.2 ml 2 M KC1, incubation at ambient temperatures for 1 h, centrifugation, washing of the lower phase with 2 ml CH3OH/10 mM KH2PO4/CHC13 (48 : 47 : 3) containing 0.25% HC1 and evaporation, the residue was dissolved in 100 ~l CHC13 [11]. Separation of the phospholipids was performed with 20 × 20 cm silica gel thin layer plates F 1500 from Schleicher and Schuell according to Ref. 12. After a prerun with CH3OH, spotting of the samples with microcaps, drying in a dessicator filled with silica gel at 4°C for 3 h, the plates were developed with CHC13/CH3OH/glacial acetic acid/0.9% NaC1 ( 5 0 : 2 5 : 8 : 2 . 5 ) at 4°C. Quantitative determination of the individual components was performed according to Ref. 13. The plates were sprayed with a solution of 0.035% 6-p-toluidino-2-naphthalenesulfonic acid in 0.1 M Tris--I-IC1 of pH 7.4, dried and the spots were scanned at Aa~s nm w i t h t h e fluorescence scanner CS-920 from Shimadzu. With each determination several different amounts of reference phospholipids were spotted on the same plate, developed and scanned, in order to establish a suitable calibration. Results and discussion
Phosphate incorporatio n in the total phospholipids As described in previous work [8] suspension cultured cells of C. roseus, after freezing and thawing twice, incorporate 32p from exogenous ATP into their phospholipid fraction due to phospholipid kinases
which obviously act in situ on endogenous lipid substrate. It was further shown [8] that the kinetics of this phosphorylation is rather fast with a short linear phase and leveling off after about 5 rain at 25°C. For the activity determination of these phospho. lipid kinases, in the present work, incubation was performed for 2 rain only to ensure linear time kinetics. In Fig. 1 cells were harvested at different times during the growth cycle and their phospholipid phosphorylation activity was determined. Incorporation into the total phospholipid fraction shows a very interesting profile with a peak activity at day 5. It has been shown [1,2] that the growth cycle of a plant suspension culture can be divided into 4 phases: after the initial lag phase and the subsequent cell division phase a further growth phase follows which is due mostly to cell elongation caused by the enlargement of the vacuole. Whereas the cell division phase is limited by the amount of the medium phosphate, the cell elongation phase is limited by the size of the carbon source [2]. When the accumulated carbon source decreases below a small threshold value, the starvation phase begins [2]. There is an exponential increase in the phospholipid phosphorylation activity (Fig. 1) in the lag and cell division phase. The decrease obviously indicates the transition to the cell elongation phase with a drop to very low activity at starvation.
Incorporation into the individual components Resolution of the phosphorylated phospholipids by thin layer chromatography reveals a very simple picture [81. The main incorporation (Fig. 2A) from ATP occurs by a phosphatidylinositol kinase which leads to monophosphorylated phosphatidylinositol (PIP) and by a diacylglycerol kinase leading to phosphatidic acid (PA). There is less phosphate incorporation into the diphosphorylated phosphatidylinositol (PIP2) and into phosphatidylinositol (PI). The latter corn-
169
A
FW Phosphotipids P-32 from
E
ATP
0 0 I A
"7
0
o 04-
c
E
04 0 cO
0
.c
v
n
II bo o
o_
-6 E
b
0
1 2
I Z.
I 6
I 8
f 10
r 12
~u a " ys
Fig. I. Phosphate incorporation from labeled A T P into the phospholipid fraction of C. roseus cells during the growth cycle. Phosphorylation was performed as described (cf. Materials and methods and Ref. 8), incubation time was 2 min. Growth cycle (,) in g fresh wt. per 80 ml medium.
pound cannot be obtained directly by the action of a kinase, it reflects however PI synthesis from previously labeled PA which occurs in the endoplasmic reticulum [14]. The phosphorylation pattern is very similar to that obtained with animal cells and exogenous ATP [15]. It is interesting that the peak activity for PA labeling occurs about 2 days before the peak activity of PIP labeling, although we cannot give an explanation for this fact at present. The apparent phospholipid phosphorylation activities of Figs. 1 and 2A were related to 100 mg fresh wt. In order to eliminate changes generated by dilution through the increase of cell mass during the growth cycle, phosphorylation activities were related
to the whole growing population (Fig. 2B). The data in Fig. 2B show that the main activities, i.e. PI kinase (PIP) and diacylglycerol kinase (PA), both increase up to day 7 which probably corresponds to the end of the cell division phase. In the subsequent elongation phase the activities, correlated to the whole population, attain lower but almost constant levels and only in the starvation phase do the activities decrease to rather low values. Obviously during cell elongation there is no net synthesis of these phospholipid kinases, although the vacuolar and plasmalemma m e m brane increase strongly in this growth phase. But it is possible that in this growth phase components for new membrane synthesis
170
(,,) ~,-(1~08) OC I
/W_-I 6
oE
~
o
•
Ot I
I
(33
0_" n LL
-\
n n
n
o
~ ~ "~ 0~
o~
I
o~: L-(I~OIB)
o~
u!~/=u!d
lO~U
•~
0
...-k~
o
8 . ~ 0 ' ~
C0
EO
!.0
~_(6uuOOl)~-(u!uJz) °~d IO~U
0~
171
are recruited by degrading other cell membranes.
measured. An evaluation of this question is possible from the data of Fig. 3, which show- the PI content during the growth cycle. Related to the whole cell population about 0.5--2 mg PI are present which corresponds to 0.6--2.3 ~ m o l PI calculated on the basis of a dioleate c o m p o u n d (MW = 863). The data of Fig. 2B show total activities for the PI kinase of up to 40 nmol • min -1. As enzyme kinetics was followed for only 2 rain and PI is present in the # m o l
PI and P A content during the growth cycle In the present work the activity of the phospholipid kinases was determined by exogenous A T P and endogenous lipid substrates. It was confirmed [8] that A T P is not limiting, the concentration of the endogenous lipid substrate, however, could be limiting, although initial rates (2 rain) were
PA
<3 d
4 -30 v
E
© co
E
3-
O co v
-20
PI
Q_ 0
L O
2_
c~
..c o_ u~
0 _c U~
-10 I
E
I
I
I
I
I
I
I
0
2
/.
6
8
10
12
days Fig. 3. Content of PI and P A in the growth cycle of C. roseus cells.The lipidswere separated and determined as described in Materials and methods. Growth cycle (=) in g fresh wt. per 80 ml medium.
172
range endogenous substrate should not be limited in this case. This estimation, however, is only correct with the assumption that the endogenous lipid substrates are freely available to the phospholipid kinases measured in situ. Figure 3 shows that the PI and PA content varies strongly during the growth phase. As these values are related to the whole cell population, changes by dilution during growth are corrected for. Hence the data reveal a strong increase in the absolute content during the lag phase and cell division phase and a decrease in the cell elongation phase. This indicates that only cell division is correlated both with high enzymatic activities (Fig. 2B) and substrate constituents (PI and PA) of the PI cycle, whereas the cell elongation phase has lower but significant enzymatic activities (Fig. 2B) and a decreasing content of PA and PI (Fig. 3). Obviously regulation mechanisms exist which not only control the degree of the enzymatic activities but also the pool sizes of the lipid substrates of the PI cycle, unless both control mechanisms are linked. Conclusions
operating PI cycle is essential for cell division also in the plant kingdom. Figure 4 illustrates the PI cycle with its phospholipid constituents only. The hydrophflic head groups split off by phospholipase C (e.g. inositol-P3 from PIP2) are not shown. There is evidence, however, that these inositol phosphates are present in growing C. roseus cell cultures (S. Helm, unpublished results). The cell elongation phase starts when the cellular phosphate pool decreases below a low threshold value provided the C~ource is not exhausted; it is characterized by a halt in nucleotide, protein and RNA synthesis, and by a halt in net increase of cytoplasmic volume [2,16]. We have also shown that protein kinase activities decrease in this phase [10] and the present work shows that the constituents of the PI cycle (PI and PA) also decrease, whereas the phospholipid kinase activities assume a lower but significant level. Obviously the PI cycle (Fig. 4) is still operating in this phase although with reduced activity and only at starvation is this cycle shut off. The total activities of the phospholipid kinases (Fig. 2B) are rather high. Incorporation of phosphate from ATP into PIP
In previous work on N i c o t i a n a [16] and Datura [2] cell cultures we showed that the cell division phase is characterized by exponential increases in the nucleotide pool, RNA and protein content, which obviously reflects the increases in net cytoplasmic volume. It has been further shown that in Murashige/Skoog [ 17] and Linsmaier/Skoog media [18] the phosphate source, i.e. the cellular phosphate pool sets a limit on the duration of the cell division phase [2,19]. Among the early enzymes synthesized in the cell division phase are protein kinases [10] and as shown with the present work also the phospholipid kinases of the PI cycle. Figure 3 further reveals that the components of this cycle, such as PA and PI also increase exponentially in the cell division phase. This in conclusion indicates that an actively
ATP
II
DAG
"°.. . . . . . . . . . . . . . . . . .
....°°"
Fig. 4. Phosphatidylinositol cycle. This shows only the phospholipid constituents; ponents determined in the present work cated in boxes. DAG diacylglycerol; [C] lipase C.
pathway the comare indiphospho-
173 attains a value o f a b o u t 40 n m o l • min -1, this is a b o u t 2% of t he total PI c o n t e n t which is p h o s p h o r y l a t e d in 1 rain. A similar value m a y be o b t ai ned f or the p h o s p h o r y l ation o f diacylglycerol, although we have no data o f the n e t c o n t e n t of this lipid. However, the high c o n t e n t of PA (Fig. 3) supports the supposition t h a t diacylglycerol is t u r n e d over very fast. In conclusion this data indicate a r ath er high rate for PI cycle at least in the cell division phase. Acknowledgements
We a r e grateful to Dr. V. Wray for linguistic advice, t o Mrs. H. Starke for typing t he m a n u s c r i p t and Mrs. C. Lippelt for preparing the graphs. This w o r k has been s u p p o r t e d b y t h e Deutsche Forschungsgemeninschaft (Wa 9 1 / 1 5 ) and t he Fonds der Chemischen Industrie. References
4 P.W. Majerus, D.B. Wilson, T.M. Connolly, T.E. Bross and E.J. Neufeld, Trends Biochem. Sci., 10 (1985) 168. 5 W.F. Boss and M.O. Massel, Biochem. Biophys. Res. Commun., 132 (1985) 1018. 6 H. Stasser, C. Hoffmann, H. Grisebach and U. Matern, Z. Naturforsch., 41c (1986) 717. 7 S. Helm and K.G. Wagner, Biochem. Biophys. Res. Commun., 134 (1986) 1175. 8 S. Helm, A. Bauleke, C. Wylegalla and K.G. Wagner, Plant Sci.,49 (1987) 159. 9 A.S. SandeUus and M. Sommarin, F E B S Lett.,
201 (1986) 282. 10 S. Helm, H. Erdmann and K.G. Wagner, Plant Sci., 42 (1985) 159. 11 I. Litosch, S.-H. Lin and J.N. Fain, J. Biol. Chem., 258 (1983) 13727. 12 M. Goppelt and K. Resch, Anal. Biochem., 140 (1984) 152. 13 M. Jones, R.W. Keenan and P. Horowitz, J. Chromatogr., 237 (1982) 522, 14 J.B. Mudd, in: P.K. Stumpf and E.E. Conn (Eds.), Biochem. Plants, Vol. 4, Academic Press, N.Y., 1980, p. 249. 15 M.E. Dunlop and R.G. Larkins, J. Biol. Chem., 259 (1984) 8407. 16 R. Meyer and K.G. Wagner, Physiol. Plant., 65 (1985) 439. 17 T. Murashige and F. Skoog, Physiol. Plant., 15
1 H.E. Street, Tissue Culture and Plant Science, Academic Press, London, 1974. 2 C. WylegaUa, R. Meyer and K.G. Wagner, Planta, 166 (1985) 446. 3 M.J. Berridge, Biochem. J., 220 (1984) 345.
(1962) 473. 18 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 19 S. Amino, T. Fujimura and A. Komamine, Physiol. Plant., 59 (1983) 393.