Association of Phosphatidylinositol 4-Kinase with the Plant Cytoskeleton.

In eukaryotic cells, phosphatidylinositol 4-hydroxy kinase and phosphatidylinositol-4-phosphate 5-hydroxy kinase are responsible for the formation of the two second messenger precursors phosphatidylinositol-4-phosphate (Ptdlns(4)P) and phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2). In plant cells, these kinases have been considered to be exclusively membrane associated, with the majority of activity residing in the inner leaflet of the plasmalemma. By sequentially extracting carrot protoplasts with the detergent Nonidet P-40 then more rigorously with Triton X-100, we were able to remove the activity of three separate plasma membrane marker enzymes and to demonstrate that a significant proportion of cellular Ptdlns 4-kinase is associated with the cytoskeleton. When only endogenous substrates were present, Nonidet P-40-permeabilized protoplasts and Nonidet P-40-extracted cytoskeletons displayed a pattern of lipid phosphorylation similar to that obtained with isolated plant membranes or permeabilized cells, whereas the Triton X-100-extracted cytoskeletons showed little or no activity. In contrast, when exogenous substrates were added, a major proportion of PtdlnsP formed was due to kinase activity associated with the cytoskeleton as well as nuclei. However, by subtracting the activity of isolated nuclei, it could be demonstrated that a significant proportion of the detergent-resistant Ptdlns kinase activity resides with the cytoskeletal fraction. These findings suggest that the pathways of polyphosphoinositide biosynthesis in plant cells should be reevaluated to take account of the cytoskeleton and that Ptdlns(4)P itself may play a unique role in modulation of plant cytoskeletal integrity and cellular signal transduction.

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate.

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.

Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells.

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Inositol(1,4,5)trisphosphate production in plant cells: an early response to salinity and hyperosmotic stress.

Salinity and hyperosmotic stress are environmental factors that severely affect the growth and development of plants. Adaptation to these stresses is known to be a complex multistep process, but a rise in cytoplasmic Ca(2+) and increased polyphosphoinositide turnover have now been identified as being amongst the early events leading to the development of tolerance. To determine whether a causal link exists between these two events we have investigated the effects of several salts and osmotic agents on levels of inositol(1, 4,5)trisphosphate (Ins(1,4,5)P(3)) in plant cells. Our data show that salts as well as osmotic agents induce a rapid and up to 15-fold increase in cellular Ins(1,4,5)P(3) levels. The increase in Ins(1,4,5)P(3) occurs in a dose-dependent manner and levels remain elevated for at least 10 min. These data indicate that increased Ins(1,4,5)P(3) production is a common response to salt and hyperosmotic stresses in plants and that it may play an important role in the processes leading to stress tolerance.

Presence of a novel form of phosphatidylinositol 4-kinase in rat liver.

Rat liver microsomes contain two distinct forms of PtdIns 4-kinase which were resolved by heparin-Sepharose chromatography. One enzyme was identified as the type II PtdIns kinase previously isolated from exocytotic vesicles. The other enzyme, however, was a novel PtdIns 4-kinase isoform with properties differing from any other PtdIns kinase so far characterized. Both kinases were recognized by a monoclonal antibody specific for type II PtdIns 4-kinase, but the novel enzyme was considerably less sensitive to inhibition by adenosine and Ca2+ than type II enzymes, and in addition was specifically inhibited by submillimolar concentrations of dithioerythritol. The presence of a novel PtdIns 4-kinase isoform in rat liver raises the question of whether this enzyme is unique for this organ or whether it has a more widespread distribution but so far has avoided detection.

ATP-dependent regulation of nuclear Ca(2+) levels in plant cells.

Localised alterations in cytoplasmic Ca(2+) levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca(2+) levels reflect the prevailing cytoplasmic Ca(2+) levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca(2+) can be transported across nuclear membranes in an ATP-dependent manner and that over 95% of Ca(2+) is accumulated into a pool releasable by the Ca(2+) ionophore A.23187. ATP-dependent nuclear Ca(2+) uptake did not occur in the presence of ADP or ADPgammaS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran-linked Indo-1 showed that the initial ATP-induced rise in [Ca(2+)] occurs in the nuclear periphery. The occurrence of ATP-dependent Ca(2+) uptake in plant nuclei suggests that alterations of intranuclear Ca(2+) levels may occur independently of cytoplasmic [Ca(2+)] changes.

The role of the actin cytoskeleton in plant cell signaling.

The plant actin cytoskeleton provides a dynamic cellular component which is involved in the maintenance of cell shape and structure. It has been demonstrated recently that the actin cytoskeleton and its associated elements provide a key target in many signaling events. In addition to acting as a target, the actin cytoskeleton can also act as a transducer of signal information. In this review we describe some newly discovered aspects of the roles of the actin cytoskeleton in plant cell signaling. In addition to a summary of the roles played by actin-binding proteins, we also briefly review the progress made in understanding how the actin cytoskeleton participates in the self-incompatibility response in pollen tubes. Finally, the emerging importance of the actin cytoskeleton in the perception and responses to stimuli such as gravity, touch and cold stress exposure are discussed. Contents I. Introduction - the actin cytoskeleton 13 II. Actin-binding proteins 14 III. The actin cytoskeleton as a target and mediator of plant cell signaling 20 IV. Summary and conclusion 25 References 25 Acknowledgements 25.

Phosphoinositides and protein phosphorylation in plant signal transduction.

A highly complex set of interactions are responsible for the perception and transduction of signals in living cells. It is likely that a number of fundamental principles of signalling mechanisms are of early evolutionary origin, have been highly conserved and are shared by apparently disparate organisms. Possible clues to the biochemical and molecular basis of plant signalling might thus be obtained from research carried out on other eukaryotes. Like mammalian cells, plant cells have been found to possess a phosphoinositide system and also make extensive use of phosphorylation and dephosphorylation cascades. The potential role of these mechanisms in plant cell signalling is reviewed.

Inositol(1,4,5)trisphosphate production in plant cells: stimulation by the venom peptides, melittin and mastoparan.

The ability of the amphiphilic peptides, melittin and mastoparan, to modulate the production of inositol(1,4,5)trisphosphate in cultured plant (Daucus carota L.) cells was investigated. When added to intact cells melittin and mastoparan caused a rapid and dose-dependent increase in inositol(1,4,5)trisphosphate concentrations. In isolated protoplasts, inositol(1,4,5)trisphosphate levels were 12- to 16-fold higher than in the corresponding cells and neither melittin nor mastoparan was able to significantly affect inositol(1,4,5)trisphosphate production. Melittin and mastoparan had a strong inhibitory effect (IC50: 20 microM) on the activity of polyphosphoinositide-specific phospholipase C in purified plasma membranes. These results demonstrate that the plant phosphoinositide system can be activated by amphiphilic peptides in a manner analogous to that observed in specialized mammalian cells but that important functional components are altered, or lost, by the disruption of the intact cell state.

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate.

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.

Identification of a phosphatidylinositol 3-hydroxy kinase in plant cells: association with the cytoskeleton.

Radiolabelling experiments have revealed that plant cells contain the two 3-phosphorylated phosphoinositides: PtdIns3P and PtdIns(3,4)P2 [Brearley and Hanke (1992) Biochem. J. 283, 255-260]. However, nothing is known about the enzymes involved in the metabolism of these plant 3-phosphorylated phosphoinositides. In this study we demonstrate the presence of a PtdIns 3-hydroxy kinase(s) in plant cells. This activity was enriched in the cytoskeletal fraction whereas only low levels of phosphoinositide 3-hydroxy kinase could be detected in plasma membranes and microsomal preparations. This cytoskeletal phosphoinositide 3-hydroxy kinase was found to be wortmannin insensitive and thus resembles PtdIns-specific 3-hydroxy kinases of which vps34p is one example.

Detection of glycolipids as biotinylated derivatives using enhanced chemiluminescence.

A new method for the detection of glycolipids as biotinylated derivatives is presented. This method is based on the detection of lipids by enhanced chemiluminescence (ECL) following conjugation with streptavidin-horseradish peroxidase (SHRP). Partial biotinylation of glycolipids is achieved after mild oxidation of the glycan moiety with Na-meta-periodate to increase the availability of biotin-reactive sites. There are several significant advantages of the glycolipid ECL detection: it avoids the need for radio-labeling; it provides the possibility of antibody probing; it allows reprobing of SHRP conjugate to adjust background and luminol light emission levels; it permits easy and accurate quantification of glycolipids at low concentrations; and it involves nondestructive staining, thereby enabling further molecular analysis.

Maize profilin isoforms are functionally distinct.

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-l-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.

Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots.

Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-(32)P]Ins(1,4,5)P(3) and [2-(3)H]Ins(1,4,5)P(3) as tracers. Ins(1,4,5)P(3) was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P(2)] whereas inositol(1,4)bisphosphate [Ins(1,4)P(2)] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P(4). Dephosphorylation of Ins(1,4,5)P(3) to Ins(4,5)P(2) was dependent on Ins(1,4,5)P(3) concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P(3) to Ins(4,5)P(2) and Ins(1,4,5,X)P(4) was inhibited by 55 micromolar Ca(2+). This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P(3) and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.

Inositol-containing lipids in suspension-cultured plant cells: an isotopic study.

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [(32)P]orthosphosphate, [2-(3)H]glycerol, and myo-[2-(3)H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [(32)P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.

Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction.

The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.