Transmembrane Signaling via Phosphatidylinositol 4,5-Bisphosphate Hydrolysis in Plants

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/ v) sodium deoxycholate, whereas in fractions enriched in insideout (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP2 phospholipase C was dependent on Ca21 with maximum activity at 10 to 100 Mm free Ca2 and half-maximal activation at 0.1 to 1 Mm free Ca2. In the presence of 10 gM Ca21, 1 to 2 mM MgCI2 or MgSO4 further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba2", Co2+, Cu2+, Mn2+, Ni2+, and Zn2+) inhibited the enzyme activity. The stimulatory effect by Mg2+ was observed also when 35 mm NaCI was included. Thus, the PIP and PIP2 phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP2 as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca2 , with maximum activity at 1 mm CaC12, and could not be further stimulated by Mg2+.animal cells, phospholipase C-catalyzed production of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol from PIP22 is a key event in the transduction of agonist-dependent signals over the plasma membrane (1,3,15). PIP2 is formed by a two-step phosphorylation of PI by PI kinase and PIP kinase, enzymes that are also present in plant plasma membranes (18,24,29). Because phospholipase C, preferentially active on PIP and PIP2, has also been identified in plasma membranes from plants (7,17,30), there is an enzymic basis for signal transduction through hydrolysis of polyphosphoinositides. However, very little is known about the mechanism operating in the plant plasma membrane, in part due to the scant knowledge about phospholipase C.In this communication, we present evidence for the localization of polyphosphoinositide phospholipase C activity in wheat (Triticum aestivum L. cv Drabant) plasma membranes to the cytoplasmic surface of the membrane. We also present a further characterization of the in vitro properties of the enzyme activity. MATERIALS AND METHODS

“…Plant phosphoinositide metabolism shows many similarities to the metabolism involved in mammalian polyphosphoinositide transmembrane signaling systems (8,12,25). In …”

Section: Materials and Methods Materialsmentioning

confidence: 99%

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.)

Pical

Sandelius²,

Melin³

et al. 1992

“…Samples containing 0.3 umol of DAG were emulsified in 0.5 mL of 0.01 M Tris buffer (pH 7.2) containing 2 gL of Triton X-l00 (Sigma) and mixed with 10 ,uL (7300 units) of the lipase. After 2 min at room temperature, the reaction was terminated by addition of 2 mL of methanol, and lipids were extracted by the procedure of Bligh and Dyer (3).…”

Section: Rhizopus Lipase Hydrolysismentioning

confidence: 99%

Diacylglycerol Metabolism in the Green Alga Dunaliella salina under Osmotic Stress

Ha¹,

Thompson²

1991

Self Cite

The sn-1,2-diacylglycerol (DAG) content and molecular species composition of Dunaliella salina whole cells and cell fractions were measured by complementary high performance liquid chromatography and gas chromatography techniques. (21) found DAG of Samanea saman pulvinus to vary from 3.5 nmol/100 nmol PL in darkacclimated tissue to 4.2 nmol/100 nmol PL following exposure to light conditions that induce PIP2 hydrolysis.The wall-less green alga Dunaliella salina, when subjected to hypoosmotic shock, responded with an immediate 50% increase in cell volume followed directly by a 30% loss of its plasma membrane-localized PIP2 (8). Concurrent responses of this sort have not been reported in other plants, but similar treatment of elasmobranch erythrocytes generated DAG whose activation of protein kinase C was postulated to trigger the restoration of normal cell volume (18). As a first step toward evaluating the possible regulatory significance ofDAG arising through PIP2 metabolism in D. salina, we have determined the molecular species composition and the subcellular disposition of the organism's endogenous DAG and quantified the osmotic stress-induced DAG changes in key membranes. The analysis, which utilizes procedures designed to quantify individual DAG molecular species in the presence of chromatographically similar neutral lipids, permits the differentiation of DAG arising from diverse metabolic origins. Making this distinction is important in view of recent reports describing the agonist-stimulated production of DAG from multiple intracellular sources (1 1, 17

“…The inositol phospholipids PIP2 and PIP2 play important roles in signal transduction (2,6,9,17). The enzymes involved in the biosynthesis of these lipids, i.e.…”

Section: Introductionmentioning

confidence: 99%

“…The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of Pi, PIP, and DAG kinase preferentially associates with actin filaments and/ or actin binding proteins and that cytoskeletal-associated Pi and PIP kinase activities can change in response to external stimulation.The inositol phospholipids PIP2 and PIP2 play important roles in signal transduction (2,6,9,17). The enzymes involved in the biosynthesis of these lipids, i.e.…”

mentioning

confidence: 99%

Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture

Tan¹,

Boss²

1992

Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of Pi, PIP, and DAG kinase preferentially associates with actin filaments and/ or actin binding proteins and that cytoskeletal-associated Pi and PIP kinase activities can change in response to external stimulation.The inositol phospholipids PIP2 and PIP2 play important roles in signal transduction (2,6,9,17). The enzymes involved in the biosynthesis of these lipids, i.e. PI 4-kinase and PI 4-monophosphate 5-kinase, have been found to exist in the plasma membrane (5, 22), cytosol (19), and nucleus (13) of plant cells. In addition to being the source of second messengers, PIP and PIP2 may be involved in regulating the organization of the cytoskeleton (12,14,16,23,25,26).It has been demonstrated in animal cells that the enzymes involved in phosphoinositide metabolism, i.e. PI, PIP, and DAG kinase activities, also are associated with the actin '