RESUMEN
⢠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.
RESUMEN
PtdIns phosphate kinases (PIPkins), which generate PtdInsP(2) isomers, have been classified into three subfamilies that differ in their substrate specificities. We demonstrate here that the previously identified AtPIP5K1 gene from Arabidopsis thaliana encodes a PIPkin with dual substrate specificity in vitro, capable of phosphorylating PtdIns3P and PtdIns4P to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) respectively. We also show that recombinant AtPIP5K1 is phosphorylated by protein kinase A and a soluble protein kinase from A. thaliana. Phosphorylation of AtPIP5K1 by protein kinase A is accompanied by a 40% inhibition of its catalytic activity. Full activity is recovered by treating phosphorylated AtPIP5K1 with alkaline phosphatase.
Asunto(s)
Arabidopsis/enzimología , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fraccionamiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genes Reporteros/genética , Técnicas In Vitro , Datos de Secuencia Molecular , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.
Asunto(s)
Arabidopsis/enzimología , Motivos EF Hand/fisiología , Fosfolipasas de Tipo C/metabolismo , Catálisis , Membrana Celular/enzimología , Secuencia Conservada , Diglicéridos/biosíntesis , Inositol 1,4,5-Trifosfato/biosíntesis , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Estructura Terciaria de Proteína/fisiología , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
In animal cells, phosphoinositides are key components of the inositol 1,4,5-trisphosphate/diacylglycerol-based 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.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Arabidopsis/citología , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Concentración Osmolar , Radioisótopos de Fósforo , Cloruro de SodioRESUMEN
Phosphatidylinositol metabolism plays a central role in signaling pathways in animals and is also believed to be of importance in signal transduction in higher plants. We report here the molecular cloning of a cDNA encoding a previously unidentified 126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4Kbeta) from the higher plant Arabidopsis thaliana. The novel protein possesses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a catalytic domain. An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of charged amino acids is specific to this plant enzyme. Recombinant AtPI4Kbeta expressed in baculovirus-infected insect (Spodoptera frugiperda) cells phosphorylated phosphatidylinositol exclusively at the D4 position of the inositol ring. Recombinant protein was maximally activated by 0.6% Triton X-100 but was inhibited by adenosine with an IC50 of approximately 200 microM. Wortmannin at a concentration of 10 microM inhibited AtPI4Kbeta activity by approximately 90%. AtPI4Kbeta transcript levels were similar in all tissues analyzed. Light or treatment with hormones or salts did not change AtPI4Kbeta transcript levels to a great extent, indicating constitutive expression of the AtPI4Kbeta gene.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/genética , Arabidopsis/enzimología , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , SpodopteraRESUMEN
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-[17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl]aminohexyl)-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-[17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl]aminohexyl)-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.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Fosfolípidos/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Anexinas/química , Anexinas/genética , Anexinas/metabolismo , Sitios de Unión/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Secuencia Conservada , Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfolipasa D/química , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteína Quinasa C/química , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
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 microM 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.
Asunto(s)
Isoenzimas/química , Isoenzimas/metabolismo , Solanum tuberosum/enzimología , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismo , Aluminio/farmacología , Secuencia de Aminoácidos , Clonación Molecular , Secuencia Conservada , ADN Complementario , Magnesio/farmacología , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Hojas de la Planta , Raíces de Plantas , Tallos de la Planta , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Solanum tuberosum/citología , Transcripción GenéticaRESUMEN
Highly purified mitochondria from potato (Solanum tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross-contamination. When the matrix and inner membrane fractions were incubated with [gamma-32P]ATP only one and three prominent phosphoproteins were detected after SDS-PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, 18, 26, 43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca(2+)-dependent protein kinase and more than 20 endogenous substrates.
Asunto(s)
Membranas Intracelulares/química , Mitocondrias/química , Fosfoproteínas/análisis , Proteínas Quinasas/análisis , Solanum tuberosum/química , Autorradiografía , Electroforesis en Gel de Poliacrilamida , Fosforilación , Proteínas de Plantas/análisisRESUMEN
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 (PIP(2)) 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 PIP(2) phospholipase C was dependent on Ca(2+) with maximum activity at 10 to 100 mum free Ca(2+) and half-maximal activation at 0.1 to 1 mum free Ca(2+). In the presence of 10 mum Ca(2+), 1 to 2 mm MgCl(2) or MgSO(4) further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+)) inhibited the enzyme activity. The stimulatory effect by Mg(2+) was observed also when 35 mm NaCl was included. Thus, the PIP and PIP(2) 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 PIP(2) as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca(2+), with maximum activity at 1 mm CaCl(2), and could not be further stimulated by Mg(2+).
RESUMEN
The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.