Cotton fiber annexins: a potential role in the regulation of callose synthase

Cotton fibers contain a characteristic set of proteins which interact with plasma membranes in a Ca²⁺-dependent manner. The association of these proteins with the membrane is correlated with a reduced level of UDP-glucose: (1→3)-β-glucan (callose) synthase activity. Analysis of the proteins released from membranes by EDTA treatment shows that the most abundant proteins comprise a family of at least three polypeptides (p34) which resemble annexins. This resemblance includes similarity in size (about 34 kDa), sequence homology, Ca²⁺-dependent precipitation or interaction with the plasma membrane, and ability to serve as a substrate for phosphorylation by endogenous protein kinase(s) which also bind to the membranes in a Ca²⁺-dependent manner. A purified fraction of these annexins binds to, and inhibits, the activity of a partially purified cotton fiber callose synthase. These findings suggest that one possible function of annexin(s) in plants is to modulate the activity and/or localization of callose synthase.     

Properties and partial protein sequence of plant annexins

We have examined the characteristics of Ca²⁺-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23. Partial sequence analysis reveals that peptides from p35 and p33 have identity to members of the annexin family of animal proteins and to annexins from tomato. Interestingly, multiple sequence alignments reveal that the domain responsible for Ca²⁺ binding in animal annexins is not conserved in these plant peptide sequences. Although p33 and p35 share the annexin characteristic of binding to membrane lipid, unlike annexins II and VI they do not associate with detergent-insoluble cytoskeletal proteins or with F-actin from either plants or animals. Immunoblotting with antiserum raised to p33/p35 from maize reveals that cross-reactive polypeptides of 33 to 35 kilodaltons are also present in protein extracts from pollen tubes of L. longiflorum. Immunolocalization at the light microscope level suggests that these proteins are predominantly confined to the nongranular zone at the tube tip, a region rich in secretory vesicles. Our hypothesis that plant annexins mediate exocytotic events is supported by the finding that p23, p33, and p35 bind to these secretory vesicles in a Ca²⁺-dependent manner.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Neuronal Localization of Ca2+-dependent Protein Phosphorylation in Brain

Abstract: The cellular localization of two Ca²⁺-dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P₂) fraction was preincubated with ³²P_j for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca²⁺. In the second system, crude synaptosomal membranes isolated from the P₂ fraction were incubated with [γ-³²P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a "calcium-dependent regulator" plus Ca²⁺. Kainic acid lesioning greatly reduced the amount of Ca-⁺-dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca²⁺-dependent protein phosphorylation systems.     

DOES COLCHICINE AFFECT AGGREGATION OF NORMAL AND TRANSFORMED BHK CELLS DIFFERENTLY?

The effect of colchicine and vinblastine on cell aggregation was studied, using BHK cells and their transformed derivatives (pyBHK cells). When cells were dissociated with EDTA and the assay was made in a Ca²⁺-containing medium, the aggregation of transformed cells was prevented by colchicine and vinblastine, whereas the aggregation of normal cells was unaffected. When a Ca²⁺-free medium was used for aggregation, neither type of cell was influenced by these drugs. BHK and pyBHK cells, dissociated by trypsin in the presence of Ca²⁺, can aggregate only in the Ca²⁺-containing medium and the aggregation of both cell types was equally prevented by colchicine and vinblastine. Based on these results, it was concluded that colchicine and vinblastine inhibited the Ca²⁺-dependent mechanism of cell adhesion, but not the Ca²⁺-independent one which occurs in the Ca²⁺-free aggregation medium.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Several compartments of Saccharomyces cerevisiae are equipped with Ca2+-ATPase(s)

Abstract Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of ⁴⁵Ca²⁺ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of ⁴⁵Ca²⁺ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca²⁺-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca²⁺-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Tyrosine Hydroxylase in "Leaky" Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP-Dependent Systems

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca²⁺, the other on cyclic AMP. The Ca²⁺-dependent system is half-maximally activated by 1-2 μ M Ca²⁺ and 0.5 m M ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 m M ) does not inhibit significantly Ca²⁺-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 μ M cyclic AMP and about 0.3 m M ATP. Ca²⁺-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10⁻⁸ M Ca²⁺ plus 2 × 10⁻⁵ M cyclic AMP or of 10⁻⁵ M Ca²⁺, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca²⁺-dependent system, whereas the other site(s) are phosphorylated by both the Ca²⁺- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.     

Inhibition of Ca2+-dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca²⁺-dependent but not the Ca²⁺-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca²⁺ using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca²⁺ influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca²⁺-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca²⁺.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Histamine-Evoked Chromaffin Cell Scinderin Redistribution, F-Actin Disassembly, and Secretion: In the Absence of Cortical F-Actin Disassembly, an Increase in Intracellular Ca2+ Fails to Trigger Exocytosis

Abstract: Histamine is a known chromaffin cell secretagogue that induces Ca²⁺-dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca²⁺ utilized in histamine-evoked secretion. Here we report that histamine-H₁ receptor activation induces redistribution of scinderin, a Ca²⁺-dependent F-actin severing protein, cortical F-actin disassembly, and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca²⁺ and were triggered by release of Ca²⁺ from intracellular stores. The trigger for the release of Ca²⁺ was inositol 1,4,5-trisphosphate because U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP₃ and intracellular Ca²⁺ levels, scinderin redistribution, cortical F-actin disassembly, and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca²⁺, blocked the rise in intracellular Ca²⁺ concentration, scinderin redistribution, F-actin disassembly, and catecholamine secretion in response to histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca²⁺ from intracellular stores. This suggests that protein kinase C is involved in histamine-induced responses. The results also show that in the absence of F-actin disassembly, rises in intracellular Ca²⁺ concentration are not by themselves capable of triggering catecholamine release.     

Ca2+-dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat ( Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [³²P]-ATP into phosphoproteins was maximal in the presence of 1 m M CaCl₂ and 5 m M MgCl₂Ca²⁺ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin-layer electrophoresis showed that the Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca²⁺ dependent protein phosphorylktion was inhibited by phenothiazine-derived drugs. Addition of S-adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca²⁺-dependent protein phosphorylation.     

Presynaptic Inhibition by Concanavalin A: Are α-Latrotoxin Receptors Involved in Action Potential-Dependent Transmitter Release?

Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [³H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 µ M . Concanavalin A also reduced the release triggered by extracellular Ca²⁺ in neurons depolarized by 25 m M K⁺ or rendered Ca²⁺-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α₂-adrenergic agonist UK 14,304. Inactivation of G_s and G_i/G_o type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K⁺ and Ca²⁺ currents. In contrast, the lectin efficiently blocked both the Ca²⁺-dependent and -independent α-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α- d -mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α-latrotoxin, concanavalin A failed to alter K⁺-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of α-latrotoxin receptors may reduce action potential-dependent transmitter release.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

From common signalling components to cell specific responses: insights from the cereal aleurone

Studies into the molecules underlying plant signal transduction events continue to reveal the involvement of highly conserved factors such as Ca²⁺, calmodulin, cyclic GMP and phospholipases in a remarkably diverse array of physiological processes. The hormonal response systems in the aleurone cells of the cereal grain and in the stomatal guard cell are beginning to reveal how diversity of response can be hard wired into these cells despite the use of these common signalling intermediates. In both the aleurone and the guard cell ABA signalling operates through the action of phospholipase D and alterations in a Ca²⁺-dependent signalling system. The role of phospholipase D is highly analogous in these two divergent cell types, perhaps reflecting the closeness of this enzyme to a conserved ABA receptor. However, specificity in response becomes evident in elements downstream from PLD, such as in the Ca²⁺ signalling system. For example, ABA has opposite effects on cytoplasmic Ca²⁺ in the aleurone and guard cell. Combining the Ca²⁺-dependent signalling activities in networks with parallel regulatory activities such as cyclic GMP appears to underlie the flexible regulatory systems that are the hallmark of plant cell function.