Self-incompatibility triggers programmed cell death in Papaver pollen

Sexual reproduction in many angiosperm plants involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding. SI is genetically controlled by the S-locus, and involves highly specific interactions during pollination between pollen and the pistil on which it lands. This results in the rejection of incompatible ('self') pollen, whereas compatible ('non-self') pollen is allowed to fertilize the plant. In Papaver rhoeas, S-proteins encoded by the stigma component of the S-locus interact with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in the inhibition of pollen-tube growth. Programmed cell death (PCD) is a mechanism used by many organisms to destroy unwanted cells in a precisely regulated manner. Here we show that PCD is triggered by SI in an S-specific manner in incompatible pollen. This provides a demonstration of a SI system using PCD, revealing a novel mechanism to prevent self-fertilization. Furthermore, our data reveal that the response is biphasic; rapid inhibition of pollen-tube growth is followed by PCD, which is involved in a later 'decision-making' phase, making inhibition irreversible.     

Aggregation of chromaffin granules by calpactin at micromolar levels of calcium

Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).     

S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility

Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell-cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.     

Modulation of dihydropyridine-sensitive Ca2+ channels by glucose metabolism in mouse pancreatic beta-cells

Glucose stimulates insulin secretion from the pancreatic beta-cell by increasing the cytosolic calcium concentration. It is believed that this increment results mainly from Ca2+ influx through dihydropyridine-sensitive calcium channels because insulin secretion is abolished by dihydropyridine antagonists and is potentiated by dihydropyridine agonists. Glucose may influence Ca2+ influx through these channels in two ways: either by regulating the beta-cell membrane potential or by biochemical modulation of the channel itself. The former mechanism is well established. Glucose metabolism, by closing ATP-sensitive K+ channels, depolarizes the beta-cell membrane and initiates Ca2+-dependent electrical activity, with higher glucose concentrations further increasing Ca2+ influx by raising the frequency of action potentials. We show here that glucose metabolism also increases calcium influx directly, by modulating the activity of dihydropyridine-sensitive Ca2+ channels.     

The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins

The self-incompatibility response in Brassica allows recognition and rejection of self-pollen by the stigmatic papillae. The transmembrane S-locus receptor kinase (SRK), a member of the receptor-like kinase superfamily in plants, mediates recognition of self-pollen on the female side, whereas the S-locus cysteine-rich protein (SCR) is the male component of the self-incompatibility response. SCR is presumably located in the pollen coat, and is thought to be the SRK ligand. Although many receptor-like kinases have been isolated in plants, the mechanisms of signal transduction mediated by these molecules remain largely unknown. Here we show that SRK is phosphorylated in vivo within one hour of self-pollination. We also show that, in vitro, autophosphorylation of SRK is prevented by the stigma thioredoxin THL1 in the absence of a ligand. This inhibition is released in a haplotype-specific manner by the addition of pollen coat proteins. Our data indicate that SRK is inhibited by thioredoxins and activated by pollen coat proteins.     

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.     

Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas.

Exocrine gland cells secrete Cl(-)-rich fluid when stimulated by neurotransmitters or hormones. This is generally ascribed to a rise in cytosolic Ca2+ concentration ([Ca2+]i), which leads to activation of Ca2(+)-dependent ion channels. A precise understanding of Cl- secretion from these cells has been hampered by a lack of knowledge about the spatial distribution of the Ca2+ signal and of the Ca2(+)-dependent ion channels in the secreting epithelial cells. We have now used the whole-cell patch-clamp method and digital imaging of [Ca2+]i to examine the response of rat pancreatic acinar cells to acetylcholine. We found a polarization of [Ca2+]i elevation and ion channel activation, and suggest that this comprises a novel 'push-pull' mechanism for unidirectional Cl- secretion. This mechanism would represent a role for cytosolic Ca2+ gradients in cellular function. The cytosolic [Ca2+]i gradients and oscillations of many other cells could have similar roles.     

Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells

Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.     

H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins

A group of calcium-binding proteins which bind to biomembranes has recently been identified in widely different cells and tissues (refs 1-7, reviewed in ref. 8). Three of these proteins (p70, p36 and p32.5) cross-react with antiserum to calelectrin, a Ca2+-binding protein (relative molecular mass 34,000 (34K] from the ray Torpedo marmorata, giving rise to their designation as calelectrin-related proteins. We now report that calelectrin, p36 and p32.5 contain a 17-amino-acid consensus sequence which is conserved and present in multiple copies. We suggest that this sequence may be common to other members of this new group of Ca2+-binding proteins and may underlie their unusual mode of combination with biomembranes.     

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.     

Role of Ubisch bodies secreted by tapetum in Ca2+ transprot

The distribution of Ca2+ in the anthers of wheat was observed using cytochemical method of potassium antimonite. At the later tetrad stage, Ubisch bodies carrying Ca2+ were observed on the inner surface of tapetum, in anther locule and on pollen surface. The Ubisch bodies contacted with pollen, and Ca2+ began to accumulate on pollen surface. At the uninucleate pollen stage, abundant Ubisch bodies were distributed in anther locule, and the amount of Ca2+ on pollen surface increased. At the mature pollen stage a large amount of Ca2+ ions were localized on the inner surface of tapetum, the surface of pollen and Ubisch bodies. In the pollen wall, Ca2+ precipitates arranged in radial lines. These results demonstrated that Ubisch bodies were involved in Ca2+ transport from anther wall to pollen surface at some developmental stages of anther.     

Two polyphosphatidylinositide metabolites control two K+ currents in a neuronal cell

Hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) produces two prospective intracellular messengers: inositol 1,4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores; and diacylglycerol (DG), which activates protein kinase C. Here we show how the formation of these two substances triggered by one external messenger, bradykinin, leads to the appearance of two different sequential membrane conductance changes in the neurone-like NG108-15 neuroblastoma-glioma hybrid cell line. In these cells bradykinin rapidly hydrolyses PtdIns(4,5)P2 to InsP3 and DG, raises intracellular Ca2+ and hyperpolarizes then depolarizes the cell membrane. By voltage-clamp recording we show that the hyperpolarization results from the activation pharmacologically-identifiable species of Ca2+-dependent K+ current. This is also activated by intracellular injections of Ca2+ or InsP3 so may be attributed to the formation and action of InsP3. The subsequent depolarization results primarily from the inhibition of a different, voltage-dependent K+ current, the M-current that is also inhibited by DG activators. Hence we describe for the first time a dual, time-dependent role for these two intracellular messengers in the control of neuronal signalling by a peptide.     

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.     

T-type calcium channel regulation by specific G-protein betagamma subunits

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.     

Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation.

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.     

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.     

Identification of the pollen determinant of S-RNase-mediated self-incompatibility

Many flowering plants have adopted self-incompatibility mechanisms to prevent inbreeding and promote out-crosses. In the Solanaceae, Rosaceae and Scrophulariaceae, two separate genes at the highly polymorphic S-locus control self-incompatibility interactions: the S-RNase gene encodes the pistil determinant and the previously unidentified S-gene encodes the pollen determinant. S-RNases interact with pollen S-allele products to inhibit the growth of self-pollen tubes in the style. Pollen-expressed F-box genes showing allelic sequence polymorphism have recently been identified near to the S-RNase gene in members of the Rosaceae and Scrophulariaceae; but until now have not been directly shown to encode the pollen determinant. Here we report the identification and characterization of PiSLF, an S-locus F-box gene of Petunia inflata (Solanaceae). We show that transformation of S1S1, S1S2 and S2S3 plants with the S2-allele of PiSLF causes breakdown of their pollen function in self-incompatibility. This breakdown of pollen function is consistent with 'competitive interaction', in which pollen carrying two different pollen S-alleles fails to function in self-incompatibility. We conclude that PiSLF encodes the pollen self-incompatibility determinant.     

Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.