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.Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Three of the five free hydroxyl groups of PtdIns can be phosphorylated in cells in different combinations. In total, seven phosphorylated derivatives of PtdIns have been detected, one of which, PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], has not been found in plant cells (Fig. 1). These inositol phospholipids are collectively referred to as phosphoinositides (PIs). In animal cells, PIs and their derivatives operate in signal transduction pathways triggered by stimuli as diverse as growth factors, hormones, neurotransmitters, and light (Berridge, 1993).Historically, the first major insight into the importance of PIs was the discovery that the two PtdIns 4,5-bisphosphate [PtdIns(4,5)P 2 ]-derived second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol promote Ca 2ϩ release from internal stores and activate protein kinase C, respectively (Berridge and Irvine, 1989; for review, see Berridge, 1993). The production of these two second messengers from PtdIns(4,5)P 2 is catalyzed by PI-specific phospholipase C (PI-PLC) isoforms. During the last decade, it has become evident that in addition to serving as precursors to Ins(1,4,5)P 3 and diacylglycerol, PIs actively participate in several other cellular processes. They have been shown to regulate the dynamics of the actin cytoskeleton through interaction with actin-binding proteins (Lassing and Lundberg, 1985; Janmey and Stossel, 1987; Brill et al., 2000), and to potentiate the activation of protein kinase C (Oh et al., 1998), PI-PLC (Bae et al., 1998; Falasca et al., 1998), and phospholipase D (Pappan et al., 1997). In addition, PIs phosphorylated at the D3-hydroxy group of the inositol head group are required for specific vesicle trafficking steps (De Camilli et al., 1996; Wurmser et al., 1999) and are able to activate the protein kinase Akt/PKB and PI-dependent kinases (Wymann and Pirola, 1998). A 3-phosphoryla...

Oscillations in cytosolic free Ca Stomata form pores in the epidermis of the leaf that allow CO 2 uptake for photosynthesis and water loss via transpiration. During drought, the loss of water through transpiration is reduced in response to an increase in the levels of the plant hormone abscisic acid (ABA) in the leaves (1). ABA stimulates the efflux of K ϩ from the guard cells that surround the stomatal pore, resulting in a reduction in guard-cell turgor and a decrease in the width of the pore (2). An increase in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) has been shown to be an early event in the signal transduction pathway by which ABA stimulates a reduction in guard-cell turgor (3-8). In addition, components of Ca 2ϩ -based second messenger systems found in animals have been identified in guard cells (9). However, little is known about the process by which the information required to describe the strength of the ABA stimulus is encoded in ABA-induced changes in guard-cell [Ca 2ϩ ] cyt or the mechanism(s) by which these changes are generated.It has been proposed that oscillations in [Ca 2ϩ ] cyt have the potential to increase the amount of information encoded by changes in [Ca 2ϩ ] cyt in plant cells through the generation of a stimulus-specific Ca 2ϩ signature (9, 10). Studies in animals suggest that signaling information may be encoded in the period and͞or the amplitude of stimulus-induced oscillations in [Ca 2ϩ

In animal cells, phosphoinositides are key components of the inositol 1,4,5-trisphosphate/diacylglycerolbased signaling pathway, but also have many other cellular functions. These lipids are also believed to fulfill similar functions in plant cells, although many details concerning the components of a plant phosphoinositide system, and their regulation are still missing. Only recently have the different phosphoinositide isomers been unambiguously identified in plant cells. Another problem that hinders the study of the function of phosphoinositides and their derivatives, as well as the regulation of their metabolism, in plant cells is the need for a homogenous, easily obtainable material, from which the extraction and purification of phospholipids is relatively easy and quantitatively reproducible. We present here a thorough characterization of the phospholipids purified from [ 32 P]orthophosphate-and myo-[2-3 H]inositol-radiolabeled Arabidopsis thaliana suspension-cultured cells. We then show that NaCl treatment induces dramatic increases in the levels of phosphatidylinositol 4,5-bisphosphate and diacylglycerol pyrophosphate and also affects the turnover of phosphatidylcholine. The increase in phosphatidylinositol 4,5-bisphosphate was also observed with a non-ionic hyperosmotic shock. In contrast, the increase in diacylglycerol pyrophosphate and the turnover of phosphatidylcholine were relatively specific to salt treatments as only minor changes in the metabolism of these two phospholipids were detected when the cells were treated with sorbitol instead of NaCl.Phosphoinositides are quantitatively minor phospholipids that play an important role in the transduction of physiological signals, such as hormones, growth factors, and neurotransmitters in animal cells (1). One of the key early events triggered by these physiological stimuli is the activation of phosphoinositide-specific phospholipase C (PI-PLC), 1 resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) to the two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and diacylglycerol, which induce Ca 2ϩ release from internal stores and stimulate protein kinase C, respectively (1, 2). During the last decade, it has become evident that in addition to serving as precursors to Ins(1,4,5)P 3 and diacylglycerol, phosphoinositides actively participate in other cellular functions: they have been shown to regulate the dynamics of the actin cytoskeleton through the interaction with actin-binding proteins (3, 4), and to potentiate the activation of protein kinase C (5) and PI-PLC (6, 7). In addition, phosphoinositides phosphorylated at the D 3 -hydroxy group of the inositol headgroup are required for specific vesicle trafficking steps (8, 9) and are able to activate the recently identified novel protein kinases Akt/PKB and phosphoinositide-dependent kinases (10). Recently, a new 3-phosphorylated phosphoinositide, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ), was identified and shown to accumulate in yeast cells ...

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 (PIP 2 ), 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 C 2 -like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP 2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP 2 -hydrolyzing activity at 10 M Ca 2؉ and were inhibited by Al 3؉ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg 2؉ . 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.

Summary• PI-PLCs synthesise the calcium releasing second messenger IP 3 . 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.• AtPLC s 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.