Journal of Experimental Botany, Vol. 54, No. 383, pp. 691-698,
February 1, 2003
© 2003 Oxford University Press
In vitro distribution and characterization of membrane-associated PLD and PI-PLC in Brassica napus
Received 30 April 2002; Accepted 10 October 2002
t
pánka 
dárová
Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, 166 28 Prague 6, Czech Republic
1 Present address: Institute of Experimental Botany, Academy of Sciences of Czech Republic, Prague 6, Czech Republic.
2 Present address: Laboratoire de Physiologie Cellulaire et Moléculaire, Université Pierre et Marie Curie,UMR 7632, Paris Cedex 05, France.
3 To whom correspondence should be addressed: Fax: +420 2 24355167. E-mail: olga.valentova{at}vscht.cz
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Abstract |
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Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 3.1.4.4) and phosphatidyl- inositol-specific phospholipase C (PIP2-PLC; PI-PLC 3.1.4.11) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP2-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLD
). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP2-dependent PLD activity shows the opposite trend when compared to PLD, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLD. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca2+, pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLD with G-proteins under in vitro conditions was not confirmed. Key words: Brassica napus, phospholipases, plasma membrane.
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Introduction |
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Phospholipids play an important role in many signalling pathways in animal and plant cells. Signalling cascades are triggered by the activation of phospholipid cleaving enzymes such as phospholipases C, D and A2. Their activities result in the formation of second messengers. It is increasingly clear that multiple lipid signalling enzymes often form complex networks that mediate a specific cellular response. PI-PLC releases InsP3 that promotes oscillations and increases in cytoplasmic Ca2+ (Staxen et al., 1999). The increase of Ca2+ may enhance PLD association with membranes, resulting in PLD activation (Wang, 2000; Zheng et al., 2000). PI-PLC also produces a diacylglycerol, a well-known effector molecule in animal cells. However, the role of DAG in plants remains unclear. Since phosphatidic acid, a product of PLD action can be dephosphorylated by phosphatidate phosphatase to DAG, both enzymes could be involved in the same signalling cascade. PLD-derived PA may also be a potent stimulator of the phosphatidylinositol 4-phosphate 5-kinase needed for the production of phosphatidylinositol 4,5-bisphosphate (PIP2). In addition to being an activator of one form of PLD and the substrate of PI-PLC, PIP2 also serves as a membrane attachment site for various proteins involved in membrane trafficking (Blatt, 2000).
Although plant PLDs represent a multiple gene family, only two biochemically distinct forms have been described in plant material and characterized by their requirements for PIP2 and Ca2+ (Wang, 2001). The physiological role of these two types still remains unclear. PIP2-independent PLD seems to be activated in the early stages of seed and plant development (Ryu et al., 1996). To date, the only study distinguishing between the two forms of PLD in the course of tissue development has been done on Arabidopsis thaliana (Fan et al., 1999) on different parts of 2-month-old plants.
Furthermore, the simultaneous investigation of both types of phospholipases, PI-PLC and PLDs, which are now believed to interplay in signal transduction, becomes very important. Activation of PLC by G-protein has been described (Arz and Grambow, 1994) while the evidence for the same mechanism for PLD is much less pronounced (Lein and Saalbach, 2001; Munnik et al., 1998)
The purpose of this study was to use previous results obtained in the laboratory on PLD (Novotná et al., 1999) and PI-PLC (Crespi et al., 1993) and to direct the new project to the common physiological role of these two enzymes in an important crop plant.
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Materials and methods |
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Plant material
Maturing rape seeds (Brassica napus L. cv. Lirajet) were collected at weekly intervals starting 34 d after flowering in three consecutive years.
Seeds were germinated at 30 °C for 12 h in the light, then 12 h at 10 °C in the dark. Subsequently, seedlings were grown in continuous light at 25 °C. For the experiments, hypocotyls and cotyledons were separated from other parts of the plants.
Subcellular fractionation
Plant material was homogenized at 4 °C with a mortar and pestle in the buffer (1:4, w/v) containing 70 mM Tris-MES (pH 8.0), 0.25 M sucrose, 3 mM EDTA, 0.2% BSA, 5 mM DTT, and protease inhibitors (0.23 mM PMSF, 0.83 mM benzamidine, 0.7 µM pepstatin, 1.1 µM leupeptin, and 77 nM aprotinin). The homogenate was filtered through the nylon cloth and centrifuged at 6000 g for 15 min at 4 °C. The supernatant was filtered through Miracloth (Calbiochem, Switzerland) and diluted (in the ratio 1:3) with suspension buffer containing 1.1 M glycerol, 10 mM Tris-Mes (pH 8.0) and protease inhibitors. After centrifugation at 100 000 g for 60 min at 4 °C, the soluble fraction was obtained and pelleted microsomal fraction was resuspended in suspension buffer.
Membrane fractions were further separated by sucrose gradient centrifugation (18–38%, w/w). Sucrose solutions were prepared in suspension buffer (10 ml of each). A preformed gradient was covered with 3 ml of the microsomal fraction (15–20 mg of protein) and centrifuged at 30 000 g overnight at 4 °C. Fractions of 2 ml were collected, diluted with suspension buffer (1:4, v/v) and centrifuged at 100 000 g for 60 min at 4 °C. Pellets were resuspended in minimal volume of suspension buffer and used immediately for the assays or stored at –70 °C.
Plasma membrane
Plasma membrane was purified from hypocotyls of seedlings by partitioning microsomes in an aqueous dextran-polyethylene glycol two-phase system (Larsson et al., 1994). Plant material was homogenized with a mortar and pestle at 4 °C in the homogenization buffer in the ratio 1:3 (w/v). Buffer contained 50 mM HEPES-NaOH, pH 7.5, 0.4 M sucrose, 0.1 M KCl., 0.1 M MgCl2, and protease inhibitors (see above). The homogenate was filtered through nylon net and centrifuged at 10 000 g for 10 min. The supernatant was filtered through Miracloth and the microsomal membranes were pelleted by centrifugation at 100 000 g for 35 min. Microsomes were resuspended in 5 mM phosphate buffer pH 7.8. From 100 g of fresh plant material about 20 ml of microsomal fraction was obtained for further plasma membrane purification.
The aqueous two-phase system was formed from Dextran T-500 (AP Biotech) and polyethylene glycol 3350, both at a final concentration of 6.1% (w/w), 0.43 mM phosphate buffer pH 7.8, 3 mM KCl, and 0.22 M sucrose. 2 g of the microsomal fraction were applied to the system (14 g), the cuvette content was mixed slowly 40 times and, after the stabilization of the system within 1–2 h at 4 °C, centrifuged at 1500 g for 5 min. The plasma membrane fraction (upper phase) was further purified by sequential partitioning against fresh lower phase prepared in another set of cuvettes. Pooled upper phases were diluted five times with 5 mM HEPES-Tris buffer pH 7.8 containing protease inhibitors (see above) and centrifuged at 100 000 g for 35 min. The purified plasma membrane pellet was resuspended in the dilution buffer and used immediately or stored at –70 °C.
Membrane marker assays
Microsomal fractions were obtained by sucrose density gradient centrifugation and enriched plasma membrane fractions were characterized by measuring the activity of marker enzymes according to procedures described earlier (Martinec et al., 2000).
Endoplasmic reticulum (ER) was detected by antimycin A insensitive NAD(P)H-dependent cytochrome c reductase, mitochondria by cytochrome c oxidase, for plasma membrane 1,3-ß-D-glucan synthase II activity was measured, and, finally, pyrophosphatase was used as a tonoplast marker.
Chlorophyll content
Acetone (80%, v/v) extracts of membrane fractions were measured at 720 nm and 652 nm.
Protein determination
Protein concentration in the samples was determined according to Bradford (1976), using bovine serum albumin as a standard.
PLD assay
PIP2-independent PLD (PLD) activity was measured spectrophotometrically using the choline oxidase/peroxidase system for determination of released choline by the procedure described earlier (Sajdok et al., 1995). PLD activity was determined for phosphatidyl- choline (PC) as a substrate in the presence of 120 mM CaCl2 and 10 mM sodium dodecyl sulphate.
PIP2-dependent PLD activity was measured radiometrically with [methyl-3H]PC or [methyl-14C]PC (Qin et al., 1997). Lipid vesicles were prepared from 3.6 µmol of phosphatidylethanolamine (PE), 0.32 µmol of phosphatidyl inositol 4,5-bisphosphate (PIP2), 0.22 µmol of PC, and 2.5 µCi of labelled PC. Phospholipids were dissolved in chloroform/methanol (2:1, v/v), evaporated under nitrogen and emulsified in 1 ml of water by 30 min sonication in a water bath. The incubation mixture contained 0.1 M MES buffer pH 6.8, 100 µM CaCl2, 80 mM KCl, 2 mM MgCl2, 0.1% Triton, 4–12 µg of proteins, and 0.4 mM lipid vesicles in a total volume 100 µl. After 30 min incubation at 30 °C the reaction was stopped by the addition of 1 ml of the chloroform/methanol mixture (2:1, v/v) and 100 µl of 1 M KCl. Radioactivity was measured in the water phase (200 µl aliquots were mixed with 3 ml of scintillation solution).
PI-PLC (PIP2-PLC) assay
Activity was estimated using radiolabelled substrate [3H-inositol] PIP2 followed by biphasic extraction of the reaction product inositol 1,4,5-trisphosphate as described earlier (Crespi et al., 1993).
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Results |
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Changes of PLDs and PI-PLC in vitro activities in the course of seed and plant development
To determine the distribution of different PLDs and PI-PLC in rape seed, protein extracts from seeds at different stages of development, dry seeds and the early stages of seedling growth were fractionated into soluble and membrane fractions and assayed for phospholipase activities.
The PIP2-independent PLD assay was based on the presence of 120 mM Ca2+, SDS, and egg PC as a substrate. PIP2-independent PLD was found both in soluble and membrane fractions of developing seeds, cotyledons and hypocotyls of seedlings and the mode of distribution differed in developing seeds and plants (Fig. 1A). The specific activities in soluble fractions were significantly higher in the early stages of both processes and dropped markedly in the later stages of development of seeds as well as in plants. Specific activities of membrane-bound forms decreased during seed maturation and increased in the hypocotyls only during the later stages of development.
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The PIP2-dependent form of PLD was assayed in the presence of 100 µM Ca2+ and the lipid vesicles were composed of PIP2, PE and PC in the absence of SDS. Under these conditions, the in vitro specific activities of the PIP2-dependent PLD were lower (200-fold) than those of the PIP2-independent PLD. PLD specific activities of membrane-associated fractions increased during seed development until day 64. There was very low activity in the mature (64 d) seeds. The specific activities of PLD in hypocotyls and cotyledons were increasing with a significant drop between 5 d and 7 d of seedling growth. The activity in the hypocotyls of young seedlings was 2-fold higher than in the cotyledons (Fig. 1B).
The PI-PLC assay used 0.05 mM Ca2+, sodium deoxycholate and [3H] PIP2 as a substrate. No soluble activity of PI-PLC was detected. The specific activity in the membrane fraction decreased with the degree of seed maturation and the course was similar to the course of PIP2-independent PLD activity. The activities in the seedlings were one order of magnitude higher than in developing seeds, increasing in hypocotyls, and decreasing in cotyledon parts (Fig. 1C).
Subcellular localization of phospholipases in seeds
To identify the membrane structures with which phospholipases are associated, the subfractionation of microsomal fraction prepared from developing seeds (48 d after flowering) was performed. The identity and purity of each membrane fraction were determined by assaying activities of appropriate marker enzymes as reported previously (Martinec et al., 2000). 1,3-ß-D-glucan synthase II and cytochrome c oxidase, the markers for plasma membrane and mitochondria, respectively were concentrated in the higher density fractions, numbers 7–10 (Fig. 2A, B). Pyrophosphatase, the tonoplast marker, was found at the lower density fractions, numbers 2–5 (Fig. 2B). Antimycin A-insensitive cytochrome c reductase, the ER marker, was found in the lower density fractions, numbers 1–3, and was not well separated from the tonoplast marker (results not shown). These subcellular fractions were assayed for PIP2-independent PLD and PIP2-dependent PLD and PI-PLC activities (Fig. 2C, D). The subcellular localization of PI-PLC and PLDs in developing seeds showed that the highest activities of both enzymes correlated well with the activity of 1,3-ß-D-glucan synthase II and cytochrome c oxidase.
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Isolation and characterization of plasma membrane vesicles
The plasma membrane vesicles used in the experiments were highly purified from 5 d hypocotyls for PLD and 9 d hypocotyls for PI-PLC. The enrichment of plasma membrane was achieved by partitioning microsomes in an aqueous dextran-polyethylene glycol two-phase system. The upper phase contained the plasma membranes with only minor contamination by intracellular membranes (Table 1). Typically, the plasma membrane fraction obtained was characterized by at least a 2.5-fold increase of 1,3-ß-D-glucan synthase II activity and a decrease of other marker enzyme activities.
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Biochemical characteristics of plasma membrane associated forms of PLDs and PI-PLC
The effect of pH on both forms of PLD and PI-PLC was investigated over a range of pH 5.0–8.0 (Fig. 3A, B, C). The PIP2-independent form exhibited an acidic pH optimum between 5.5 and 6.0. The PI-PLC and the PIP2-dependent PLD were most active at pH between 6.5 and 7.0.
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To determine the influence of divalent cations on phospholipases activities, free Ca2+ and Mg2+ in the reaction mixture were controlled using Ca2+/Mg2+- EGTA buffers. The activities of both enzymes were undetectable in the absence of Ca2+. The stimulation was observed at a micromolar level for the PI-PLC and the PIP2-dependent PLD activity (Fig. 4A, B). The sharp increase of PIP2-independent activity was at millimolar concentrations of Ca2+ (Fig. 4C). Mg2+caused no stimulation of PI-PLC at any concentration tested, a slight decrease was observed for the concentrations of Mg2+above 0.2 mM (data not shown).
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G-protein activation of phospholipases
To investigate whether GTPase is directly involved in the regulation of phospholipase activities, the effects of GTP-
-S, a non-hydrolysable analogue of GTP was used. For that purpose the microsomal fraction vesicles and inside-out plasma membrane vesicles were prepared by four times repeated freezing and thawing (DeHahn et al., 1997). PLD activities were, in this case, measured using the PC substrate in the form of liposomes prepared by sonication without adding the detergents (Novotná et al., 1999). Under these conditions very low PLD activity was detected on the plasma membrane and a very slight increase of about 10% was observed (Fig. 5). The effect was eliminated by the addition of a small amount of SDS (0.5 mM). No effect GTP--S was observed for PIP2-dependent PLD and PI-PLC (data not shown).
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Discussion |
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In the present study, two types of phospholipid-degrading enzyme activity, PLD and PI-PLC were followed during the development of different tissues, i.e. seeds and plants. Two types of PLD activity, differing in the demands for calcium concentration and effector molecule PIP2, were screened during these processes. These results indicate that both PLD forms exist in the soluble form and are linked to the membranes, whereas PI-PLC is exclusively associated with a membrane fraction. At the PLD activity level, one major difference is that the specific PIP2- independent in vitro activity was much higher than specific PIP2-dependent activity. This result is consistent with a recent report that demonstrated the distribution of PLDs in Arabidopsis (Fan et al., 1999). Another major difference is that the only specific PIP2-dependent PLD activity of membrane associated enzyme increased 5-fold during seed development while the PIP2- independent PLD activity decreased in both soluble and membrane fractions. Very low PIP2-dependent PLD activity was detected in dry seeds. The appearance of PIP2-independent PLD activity in the cytosol of mature dry seeds could be due to better disruption of the tissue when homogenizing dry seeds. Ryu et al. (1996) obtained similar results for extracts from soybean seeds. These authors concluded that PIP2- independent PLD activity was highest during the early and middle stages of seed development and then declined about 4-fold. In mature dry seeds, specific activity was also increasing, but no PLD mRNA was detected, suggesting that PLD activity comes primarily from the pre-existing PLD rather than de novo synthesis.
Compared to seed development, the specific activity of soluble PIP2-independent enzyme in hypocotyl and cotyledon parts of developing plants, decreased rapidly within 7 d, while specific activity of the membrane-bound form began to increase at this point. One of the possible explanations is that an increase in specific activity of the membrane-bound PIP2-independent enzyme was due to the migration of PLD from the cytosol towards the membrane. This tendency was more pronounced in hypocotyls and thus this form of PLD can play a role during rapid tissue development. This sort of redistribution of the soluble (vacuolar) form to the plasma membrane was described for castor bean fully matured leaves compared to 6 d hypocotyls (Xu et al., 1996), but not within one tissue.
The presence of PLD in the cytosol suggests that there must be mechanisms to regulate the cytoplasmic PLD activity and an important regulator of PLD is the change in cytoplasmic Ca2+concentration. The calcium-binding domain of PLD was demonstrated to be responsible for the association of the enzyme to the membrane, via an increase in free cellular Ca2+ levels (Nalefski et al., 1994).
To determine the subcellular origin of the membrane vesicles that possessed phospholipase activity, the crude microsomal membrane preparation was separated using sucrose density gradient centrifugation. Fractionation results showed that PI-PLC and PIP2-dependent PLD are associated predominantly with the plasma membrane fraction, which contained a small portion of mitochondrial membranes. PIP2-independent PLD activity was found also in the low-density fractions. These results are in a good agreement with those obtained by Xu et al. (1996), demonstrating the intracellular localization of PIP2-independent PLD in castor bean. Partitioning of microsomal membranes in an aqueous dextran–polyethylene glycol two-phase system is a standard technique for purifying plasma membrane fractions from plant tissues (Larsson et al., 1994). During isolation, the plasma membrane vesicles preferentially partition into the polyethylene glycol-rich upper phase, while all contaminating intracellular membranes partition preferentially at the interface or into the lower phase. In the present study, the relative abundance of plasma membrane vesicles in the phase-purified membrane fraction was determined by measuring the activity of 1,3-ß-D-glucan synthase II. The presence of contaminating inner membranes in the plasma membrane preparation was identified using marker enzymes and chlorophyll content. The specific activities of markers as well as chlorophyll content are significantly lower than in the corresponding residual inner membrane fraction.
The plasma membrane-associated phospholipases from rape seedlings exhibit distinctive features. Ca2+ ions are required for the activities of both enzymes. Unlike the PIP2-independent PLD, whose maximal activity in vitro requires millimolar concentrations of Ca2+, the PIP2-dependent PLD and PI-PLC are fully active at micromolar concentrations of Ca2+ and, moreover, require PIP2 for activity. The PIP2-independent PLD was most active at a pH between 5.5 and 6, whereas PIP2-dependent PLD and PI-PLC were both most active in the pH range between 6.5 and 7.0. These results are consistent with the identification and characterization of PIP2-dependent PLD activity in Arabidopsis (Pappan et al., 1997) and PI-PLC activity in Triticum aestivum (Arz and Grambow, 1994). These differences suggest that changes in the cellular levels of Ca2+, PIP2, and pH and in membrane composition, could be an important regulator that differentially activates both enzymes.
The association of phospholipases with the plasma membrane is consistent with the proposed role of phospholipases in transmembrane signalling and appears to be a potential target for the regulation by heterotrimeric G-proteins. Experiments with GTP--S have suggested that plant PLD can be G-protein-regulated (Munnik et al., 1998,1995) and Lein and Saalbach (2001) have reported the first indication for a direct interaction between PIP2-independent PLD and the G-protein -subunit in plants. Recent studies have demonstrated that GTP--S increases PIP2-independent PLD and PI-PLC activity in plasma membrane about 3–4-fold in oat cells (Park et al., 1996) and 3-fold in light-grown wheat, respectively (Arz and Grambow, 1994). In our experiments such a high increase of the PIP2-independent PLD was not achieved, probably due to the experimental conditions.
In summary, the basic knowledge of phospholipases concerning changes of their activities during the developmental processes of plant tissues, their localization and characteristics will serve as a starting point for further investigation of their common physiological role.
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Acknowledgements |
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This work was supported by the Czech Grant Agency, project no. 522/00/1332 and the Ministry of Education of the Czech Republic, project no LN00A081.
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References |
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