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