Explore the vast range of titles available.

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.

Oscillations in cytosolic free Ca2+ concentration ([Ca2+]cyt) are an important component of Ca2+-based signal transduction pathways. This fact has led us to investigate whether oscillations in [Ca2+]cyt are involved in the response of stomatal guard cells to the plant hormone abscisic acid (ABA). We show that ABA induces oscillations in guard-cell [Ca2+]cyt. The pattern of the oscillations depended on the ABA concentration and correlated with the final stomatal aperture. We examined the mechanism by which ABA generates oscillations in guard-cell [Ca2+]cyt by using 1-(6-([17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino)hexyl)-1H-pyrrole 2,5-dione (U-73122), an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC)-dependent processes in animals. U-73122 inhibited the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant PI-PLC, isolated from a guard-cell-enriched cDNA library, in a dose-dependent manner. This result confirms that U-73122 is an inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced oscillations in [Ca2+]cyt and stomatal closure. In contrast, U-73122 did not inhibit external Ca2+-induced oscillations in guard-cell [Ca2+]cyt and stomatal closure. Furthermore, there was no effect of the inactive analogue 1-(6-([17 beta- 3-methoxyestra-1,3,5(10)-trien-17-yl] amino)hexyl)-2,5-pyrrolidinedione on recombinant PI-PLC activity or ABA-induced and external Ca2+-induced oscillations in [Ca2+]cyt and stomatal closure. This lack of effect suggests that the effects of U-73122 in guard cells are the result of inhibition of PI-PLC and not a consequence of nonspecific effects. Taken together, our data suggest a role for PI-PLC in the generation of ABA-induced oscillations in [Ca2+]cyt and point toward the involvement of oscillations in [Ca2+]cyt in the maintenance of stomatal aperture by ABA

• PI-PLCs synthesise the calcium releasing second messenger IP3. We investigated the expression patterns of the Arabidopsis PI-PLC gene family and measured in vitro activity of encoded enzymes. • Gene specific RT-PCR and promoter-GUS fusions were used to analyse AtPLC gene expression patterns. The five available AtPLC cDNAs were expressed as fusion proteins in Escherichia coli. • All members of the AtPLC gene family were expressed in multiple organs of the plant. AtPLC1, and AtPLC5 expression was localized to the vascular cells of roots and leaves with AtPLC5::GUS also detected in the guard cells. AtPLC4::GUS was detected in pollen and cells of the stigma surface. In seedlings, AtPLC2 and AtPLC3 were constitutively expressed, while AtPLCs 1, 4 and 5 were induced by abiotic stresses. AtPLC1-5 were all shown to have phospholipase C activity in the presence of calcium ions. • AtPLCs showed limited tissue specific expression and expression of at least three genes was increased by abiotic stress. The differing calcium sensitivities of recombinant AtPLC protein activities may provide a mechanism for generating calcium signatures.

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 micromolar Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner

Stomatal guard cells have proven to be an attractive system for dissecting the mechanisms of stimulus-response coupling in plants. In this review we focus on the intracellular signal transduction pathways by which extracellular signals bring about closure and opening of the stomatal pore. It is proposed that guard cell signal transduction pathways may be organized into functional arrays or signalling cassettes that contain elements common to a number of converging signalling pathways. The purpose of these signalling cassettes may be to funnel extracellular signals down onto the ion transporters that control the fluxes of ions that underlie stomatal movements. Evidence is emerging that specificity in guard cell signalling may be, in part, encoded in complex spatio-temporal patterns of increases in the concentration of cytosolic-free calcium ([Ca2+]cyt). It is suggested that oscillations in [Ca2+]cyt may generate calcium signatures that encode information concerning the stimulus type and strength. New evidence is presented that suggests that these calcium signatures may integrate information when many stimuli are present.

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

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 inside-out (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 Ca2+ with maximum activity at 10 to 100 μM free Ca2+ and half-maximal activation at 0.1 to 1 μM free Ca2+. In the presence of 10 μM Ca2+, 1 to 2 mM MgCl2 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 NaCl 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 CaCl2, and could not be further stimulated by Mg2+.

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 inside-out (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 Ca2+ with maximum activity at 10 to 100 micromolar free Ca2+ and half-maximal activation at 0.1 to 1 micromolars free Ca2+. In the presence of 10 micromolars Ca2+, 1 to 2 mM MgCl2 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 NaCl 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 CaCl2, and could not be further stimulated by Mg2+