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

Abstract

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

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

“…2) and thus give access to the phosphoinositide substrate. Mg 2ϩ ions are known to activate PI-PLC activity in the presence of 10 m Ca 2ϩ in wheat plasma membranes (Pical et al, 1992;Jones and Kochian, 1995). In contrast, Al 3ϩ is a strong inhibitor of PI-PLC activity in preparations of plasma membranes from wheat roots (Jones and Kochian, 1995).…”

“…The PI-PLC assay was performed according to the method of Melin et al (1992) and Drøbak et al (1994). The reaction mixture contained 50 mm Tris/maleate, pH 6.25, 0.2 mm of 3 H-head-group-labeled PI or PIP 2 at approxi-mately 5000 dpm nmol Ϫ1 (Amersham) and varying amounts of Ca 2ϩ in a volume of 50 L. A 0.6 mm micellar lipid stock solution was prepared by mixing unlabeled (PI, PIP 2 ; Sigma) and 3 H-labeled lipid in chloroform:methanol (2:1, v/v), evaporation of solvent under a stream of nitrogen, addition of 166 mm Tris/maleate, pH 6.25, and sonication for 10 min.…”

“…The reaction was stopped by the addition of 1 mL of chloroform:methanol (2:1, v/v). Phases were separated by adding 0.25 mL of 1 n HCl, and liquid scintillation counting of water-soluble reaction products was carried out as described previously (Melin et al, 1992).…”

Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C

“…PI-PLC activity was measured as described (36). The reaction mixture contained 50 mM Tris-maleate (pH 6.0-6.5), 0.2 mM phosphatidyl[2-3 H]inositol-4,5-bisphosphate (1,000-2,000 dpm͞nmol) in micellar solution, 10 M free Ca 2ϩ , 80 mM KCl, 1.5 mM MgCl 2 , and 0.1-0.5 g of recombinant protein.…”

Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

“…PLC is a family of isoenzymes that has been classified into three groups: ␤, ␥, and ␦ (Rhee and Bae, 1997). In plants several studies have reported the biochemical presence of this enzyme (McMurray and Irvine, 1988;Tate et al, 1989;Melin et al, 1992;Pical et al, 1992;Yotsushima et al, 1992Yotsushima et al, , 1993Huang et al, 1995;De Los Santos-Briones et al, 1997). Different genes for PLC have also been cloned (Hirayama et al, 1995(Hirayama et al, , 1997Shi et al, 1995;Yamamoto et al, 1995;Kopka et al, 1998), all of them resembling the ␦ type.…”