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1.
Takamitsu Kurusu Jumpei Hamada Haruyasu Hamada Shigeru Hanamata Kazuyuki Kuchitsu 《Plant signaling & behavior》2010,5(8):1045-1047
Cytosolic free Ca2+ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca2+ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa2+ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca2+ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca2+ concentration.1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca2+ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca2+ sensors of animals.3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).5 The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,8 followed by a form of programmed cell death known as hypersensitive response (HR).9,10 Because complexed spatiotemporal patterns of cytosolic free Ca2+ concentration ([Ca2+]cyt) have been suggested to play pivotal roles in defense signaling,1,9 multiple Ca2+ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)14–19 has been suggested, other Ca2+-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity. 相似文献
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Weiss N 《Channels (Austin, Tex.)》2010,4(6):431-433
Depolarization-evoked synaptic transmission relies on the Ca2+-regulated release of quantal packets of neurotransmitters following the fusion of synaptic vesicles with the presynaptic plasma membrane. It is well known that neuronal voltage-gated Ca2+ channels (VGCC), mainly of the CaV2.1 and CaV2.2 subtypes, play a key role in the first steps of this process, by controlling extracellular Ca2+ influx into active zones of the synapse. These channels are in close association with the vesicle machinery and interact with several members of SNARE proteins (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) including syntaxin 1A/1B and SNA P-25 (Q-SNARE s), and synaptotagmin 1 and synaptobrevin 2 (R-SNARE s) (reviewed in ref. 1). All bind to the synprint (synaptic protein interaction) motif within the intracellular II -III linker of CaV2.1 and CaV2.2 channels and are responsible for a bidirectional coupling (i) linking the Ca2+ influx with the synaptic vesicle release machinery, which is essential for efficient, fast and spatially delimited neurotransmitter release2 and (ii) providing regulation of Ca2+ channel activity and thus of Ca2+ influx.3Key words: calcium channel, CaV2.1 channel, P/Q channel, syntaxin, synaptotagmin, SNAP25, exocytosis, synaptic transmissionSeveral studies have proposed that synaptotagmin 1 is the Ca2+ sensor for release, linking Ca2+ influx to vesicle fusion (reviewed in ref. 4). Synaptotagmin 1 has two repeating domains that are rich in negative charges (C2A and C2B), each capable of binding Ca2+ ions. It is commonly thought that following Ca2+ entry through VGCCs, Ca2+ ions bind to C2A and C2B domains, allowing insertion of the Ca2+ binding loops of C2A domain in the target bilayer. This then pins the vesicle to the plasma membrane to trigger exocytotic fusion. This view was supported by a point mutation in the C2A domain of synaptotagmin 1 that caused a decrease in Ca2+ affinity with a concomitant decrease of neurotransmitter release.5 However, despite the fact that synaptotagmin 1 represents the most popular candidate for Ca2+ sensor, the initial Ca2+ binding event, which occurs during the dynamic process of release is at the EEEE locus within the Ca2+ channel itself. This makes the Ca2+ channel an excellent candidate for serving as a Ca2+ sensor of secretion.6Over the past few years, the group of Daphne Atlas has performed extensive studies to differentiate the role of Ca2+ binding at the pore of the channel from Ca2+ binding to intracellular proteins during evoked-neurotransmitter release. Substituting extracellular Ca2+ by lanthanum (La3+), a trivalent cation that effectively binds to the EEEE locus of VGCCs but is unable to permeate through the channel, is sufficient to support depolarization-evoked release of catecholamine in PC12 and primary chromaffin cells, as well as insulin release in pancreatic and insulinoma cells. These results led to the suggestion that evoked release may be dependent on ion channel pore occupancy as opposed to cation influx and elevation of intracellular Ca2+ concentration.7–9 This model was further supported by experiments in which depolarization-evoked secretion of catecholamine in chromaffin cells was supported by Ca2+ bound at the selectivity filter of a non-conducting CaV1.2 channel.10 These studies are consistent with the proposal that conformational changes subsequent to Ca2+ binding at the selectivity filter of the channel are the primary trigger of secretion, whereas synaptotagmin 1 is associated with the channel and acts as a vesicle docking protein (reviewed in ref. 11).In a recent issue of Channels, Cohen-Kutner et al. extended this concept to the neuronal CaV2.1 channel.12 Using the two-electrode voltage-clamp technique on BAPTA-injected Xenopus oocyte expressing the human CaV2.1 channel (in combination with β3 and α2δ auxiliary subunits), the authors show that overexpression of syntaxin 1A (Stx1A) depresses whole-cell inward barium (Ba2+) current in a dose-dependent manner (Fig. 1, reviewed in ref. 12). As previously reported by Bezprozvanny et al.3 this effect is mainly due to a hyperpolarized shift of the steady-state inactivation curve, which decreases the number of available channels at typical resting membrane potentials. A recovery of channel activity is observed following co-expression of botulinium neurotoxin C1 (BoNT/C1) (Fig. 3, reviewed in ref. 12). In contrast, expression of the other Q-SNARE protein SNAP-25 drastically increases inward Ba2+ current (Fig. 2, reviewed in ref. 12). However, when both Q-SNARE proteins are co-expressed, CaV2.1 channel recovers wild-type P/Q kinetics and current amplitude (Fig. 2, reviewed in ref. 12). Similarly, increases in P/Q currents by expressing the R-SNARE synaptobrevin (VAMP-2) are reversed by the Q-SNARE proteins (Fig. 4, reviewed in ref. 12). Taken together these results suggest that: (i) when expressed in BAPTA injected Xenopus oocyte, each of the SNARE proteins is able to modulate the kinetic properties of CaV2.1 channel and (ii) when co-expressed, SNARE proteins no longer affect channel activity but rather form a Ca2+-independent excitosome complex with a fully functional channel. These data fit nicely with previous work from the Catterall laboratory on P/Q-type channels,13 and with previous work on N-type channels.14To investigate the relevance of CaV2.1 channel interaction with SNARE proteins for depolarization-evoked secretion, membrane capacitance changes induced in Xenopus oocytes were monitored in the presence of extracellular Ca2+, as previously shown for CaV1.2 and CaV2.2.15 While expression of CaV2.1 alone in this reconstituted release assay produced only a small change in capacitance, coexpression with the SNARE proteins efficiently induced a BoNT/C- and BoNT/A-sensitive membrane fusion, particularly when all SNARE proteins were co-expressed, i.e., when all members of the excitosome complex are present (Fig. 5, reviewed in ref. 12). Hence, increasing the amount of excitosome promotes the capability of CaV2.1 channels to produce evoked-secretion, probably by increasing the number of functional excitosome complexes (Fig. 6, reviewed in ref. 12).In summary, Cohen-Kutner et al. provide evidence that when expressed in Xenopus oocyte (and possibly in other cellular systems), CaV2.1 channels could associate with SNARE proteins at resting intracellular Ca2+ concentrations, resulting in tethering the vesicle to the channel and thereby generating docked but non-releasable vesicles. Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca2+-binding to Syt1 C2 domains. The fusion of releasable vesicles requires a conformational change of the complex that occurs within the channel itself, during an incoming action potential (Fig. 1).Open in a separate windowFigure 1A putative model of functional coupling between CaV2.1 channel and vesicle release machinery. At resting membrane potential, CaV2.1 channel associates with SNARE proteins to form an excitosome complex, in turn generating docked but non-releasable vesicle (A). Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca2+-binding to Synaptotagmin 1 C2 domains (B). The fusion of the releasable vesicle requires a conformational change of the excitosome complex that occurs within the channel itself, during an incoming action potential (C).The concept that CaV2.1 channels, besides sustaining Ca2+ influx, could also work as a molecular on/off-switch of secretion by controlling the ultimate stage of the process (i.e., the conformational change of the releasing complex) is intriguing and is worthy of further investigation. To better dissociate secretion events linked to Ca2+ entry through CaV2.1 channel from those induced by conformational changes of the channel, it would be necessary to measure secretion in the presence of a non-permeant cation such as La3+. Furthermore, one would also need to evaluate mediation of secretion by a non-conducting CaV2.1 channel, as already done for L-type channels (CaV1.2).7,9,10 Moreover, the possibility that CaV2.1 channels could control secretion via a conformational change of the releasing complex raises questions concerning the preferential channel-gating mode controlling this process. It was recently shown that application of the gating modifier BayK 8644 to non-conducting CaV1.2 channels modifies secretion kinetics of catecholamine in chromaffin cells.16 It is also well known that the auxiliary β-subunit of VGCCs modulates CaV2.1 gating modes.17 Therefore, comparing secretion mediated by a non-conducting CaV2.1 channel in the presence of different types of β-subunits would provide important information on the molecular mechanisms through which CaV2.1 channels control evoked-secretion, both at the fundamental and physiopathological levels.In conclusion, since the pioneering work by Katz and Miledi in 1967 on the importance of the extracellular Ca2+ in the “electro-secretory” process,18 the identification of the calcium channel as the Ca2+ sensor of secretion is one of the most recent and exciting steps that have been made in the understanding of the molecular aspects of the mechanisms involved in the control of depolarization-evoked neurotransmitter release. 相似文献
8.
A role for SR proteins in plant stress responses 总被引:1,自引:0,他引:1
Paula Duque 《Plant signaling & behavior》2011,6(1):49-54
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Alain Mareck Romain Lamour Annick Schaumann Philippe Chan Azeddine Driouich Jér?me Pelloux Patrice Lerouge 《Plant signaling & behavior》2012,7(1):59-61
Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.1 Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca2+ ions during their intracellular transport.2 The highly methylesterified pectins are then secreted into the apoplasm3 and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca2+ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.4 PMEs belong to a large multigene family. Sixtysix PME-related genes are predicted in the Arabidopsis genome.1 Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.5 In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins. 相似文献
12.
Calcium ion is considered a ubiquitous second messenger in all eukaryotic cells. Analysis of intracellular Ca2+ concentration dynamics has demonstrated its signalling role in plant cells in response to a wide array of environmental cues. The implication of Ca2+ in the early steps of the arbuscular mycorrhizal symbiosis has been frequently claimed, mainly by analogy with what firmly demonstrated in the rhizobium-legume symbiosis. We recently documented transient Ca2+ changes in plant cells challenged with diffusible molecules released by arbuscular mycorrhizal fungi. Ca2+ measurements by the recombinant aequorin method provided new insights into the molecular communications between plants and these beneficial fungi.Key words: legume symbioses, arbuscular mycorrhiza, calcium signalling, fungal signal, plant cell cultures, aequorinIn the rhizosphere plants meet a wide array of microorganisms. In favorable interactions, such as arbuscular mycorrhizal (AM) and nitrogen fixing symbioses, a dialogue is progressively established between the two interacting organisms to make the appropriate partner choice. These two-way communications rely on the interchange of signals released by both potential symbionts. After perception of the signalling molecules, a signal transduction pathway is induced, leading to the activation of the proper genetic and developmental program in both partners.Variations in intracellular free Ca2+ concentration occur as one of the initial steps in signalling pathways activated in plants when they encounter pathogens,1 fungal biocontrol agents2 and nitrogen-fixing bacteria.3 Molecules secreted by microorganisms, after binding to specific receptors, trigger in plant cells transient changes in cytosolic Ca2+ level, due to the influx of the ion from the extracellular environment and/or the release from internal Ca2+ storage compartments.4,5 Ca2+ messages delivered to plant cells are at least partly deciphered on the basis of their spatial and temporal features. The occurrence of different Ca2+ signatures guarantees the specificity of the ensuing physiological responses.In the legume-rhizobium symbiosis a definite pattern of Ca2+ oscillations has been reported to occur in response to the rhizobial signalling molecule, the Nod factor, in the nucleus and perinuclear cytoplasm of the root hair.6 The Ca2+ spike number has been recently demonstrated to regulate nodulation gene expression.7Legumes are able to engage in a dual symbiotic interaction, with rhizobia and AM fungi. Components of the Ca2+-mediated signalling pathway are shared by the two symbioses.8 In the mycorrhizal signal transduction pathway the involvement of Ca2+ has long been speculated, based on the observed similarities with symbiotic nitrogen fixation.3To evaluate the possible participation of Ca2+ in the early steps of the AM symbiosis, we have used a simplified experimental system given by plant cell suspension cultures stably expressing the bioluminescent Ca2+-sensitive reporter aequorin.9 The use of cultured cells circumvents the problem posed by multilayered organs: in aequorin-transformed seedlings, possible Ca2+ changes occurring in rhizodermal cells—the first place where the AM fungal signals are perceived and transduced—can be misrecorded due to luminescence calibration over all root cell layers, resulting in an underestimation of the Ca2+ signal in the responsive cells. An experimental design based on challenging host plant cells with the culture medium of different AM fungi (Gigaspora margarita, Glomus mosseae and intraradices) provided the first firm evidence that Ca2+ is involved as intracellular messenger during mycorrhizal signalling, at least in a pre-contact stage. Cytosolic Ca2+ changes, characterized by specific kinetic parameters, were triggered by diffusates obtained from AM resting and germinating spores,9 and extraradical mycelium.10 Cultured plant cells demonstrated to be competent to perceive the diffusible signal released by AM fungi and to decode the message in a Ca2+-dependent pathway. Based on these experiments, it seems that AM fungi announce their presence to the plant through the constitutive release of a chemical signal, even before experiencing the proximity of the plant or its AM symbiotic signals. The notion that the secreted fungal molecules herald, through Ca2+, a beneficial message which can be acknowledged only by competent receivers, is supported by: (1) the lack of defense response induction and the upregulation of some genes essential for the AM symbiosis initiation in host plant cells; (2) the unresponsiveness of cultured cells from the nonhost plant Arabidopsis thaliana.Ca2+-mediated perception of both AM fungal and rhizobial signals by plant cells unifies the signalling pathways activated in the two symbioses. However, the actual occurrence of Ca2+ spiking in AM symbiosis remains to be ascertained, due to limitations of the recombinant aequorin method, when applied to an asynchronous cell population. Contribution of internal Ca2+ stores, in particular the nucleus, to the observed Ca2+ changes will be a future research goal to be achieved through a pharmacological approach and/or targeting of Ca2+ indicators to intracellular compartments.The identification of the plant-derived mycorrhizal signal as strigolactones11 and their inducing activity on AM fungi12 have represented a major breakthrough in the AM symbiosis research field. Elucidation of the chemical nature of the AM fungal factor, which plays several effects on host plants,9,13–15 is eagerly awaited.Understanding how AM fungi and rhizobia select compatible plant hosts, thus activating the appropriate symbiotic program, is another facet to be considered in the future to get a complete overview of early signaling events in legume symbioses. Analysis of Ca2+ signalling implication in the microbial partner would require the delivery of reliable and sensitive Ca2+ probes (such as aequorinor GFP-based16) for Ca2+ measurements in living microorganisms. The recombinant aequorin method has been successfully applied to monitor dynamic changes in intracellular Ca2+ levels in the bacteria Anabaena sp.,17 E. coli,18 and recently by us in rhizobial strains.19 Unfortunately, AM fungi have proved not to be amenable to stable transformation, being coenocytic, multinucleate and heterokaryotic,20,21 and only transient transformants have been obtained so far.22,23 Further development of the transformation technologies may provide in the future a valuable tool to analyse, from the fungal side, signal perception and transduction during arbuscular mycorrhiza establishment. 相似文献
13.
Jennifer C Mortimer Anuphon Laohavisit Henk Miedema Julia M Davies 《Plant signaling & behavior》2008,3(9):698-699
The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca2+ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca2+ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca2+ influx, suggesting that these enzymes are involved in sensing Ca2+ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca2+ ([Ca2+]cyt) encodes plant cell signals.1 Reactive oxygen species (ROS) are potent regulators of the PM Ca2+ channels implicated in signalling and developmental increases in [Ca2+]cyt.1,2 Plasma membrane (PM) voltage (Vm) also plays a significant part in generating specific [Ca2+]cyt elevations through the opening of voltage-gated Ca2+-permeable channels, allowing Ca2+ influx.1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca2+ channels (HACCs),4 ROS-activated HACCs5 and depolarisation-activated Ca2+ channels (DACCs).6 The DACC characterisation pointed to the presence of a Cl−-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.6 This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.7 Previous studies have shown that root hair PM also harbors K+ channels (mediating inward or outward flux)8–10 and a H+-ATPase.11 A key problem to address now is how these transporters interact to generate and be influenced by PM Vm, thus gating and in turn being regulated by their companion Ca2+ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between Vm and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.12 In the “pump state” the H+-ATPase predominates, there is net H+ efflux and the hyperpolarized Vm is negative of the equilibrium potential for K+ (EK). In the “K state”, K+ permeability predominates but there is still net H+ efflux and Vm = EK. In the third state, there is net H+ influx and Vm > EK. In this depolarized H+-influx state, the H+-ATPase is thought to be inactive. Oscillations in PM Vm and H+ flux may be more profound in growing cells13,14 and oscillations between these states may explain the temporal changes in H+ flux recently observed at the apex of growing Arabidopsis root hairs.15 Peaks of H+ influx may reflect a depolarized Vm that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC4–6 and because resting Vm is usually found to be hyperpolarized. In a growing cell, with a Vm oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca2+ influx it permits could be amplified through intracellular release.The PM H+-ATPase traditionally lies at the core of models of voltage and ionic flux14,16 but in terms of [Ca2+]cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.5 AtrbohC is implicated in the transition to polar growth at normal extracellular pH5 and also osmoregulation.17 NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e− efflux systems,18 their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.19 Two H+ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e− efflux by animal NADPH oxidases is fairly constant over negative Vm and decreases at very depolarized Vm.20 AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H+ flux oscillations.15 The latter suggests that AtrbohC function does in some way affect Vm, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.21NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency22,23 to signal stress conditions. Blockade of PM Ca2+ channels by lanthanides increases superoxide production in tobacco suspension cells.24 This suggests that NADPH oxidases are involved in sensing the cell''s Ca2+ status and the prediction would be that extracellular Ca2+ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).25,26 Lowering extracellular Ca2+ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca2+ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca2+]cyt27 (Fig. 1). However, reduction of Ca2+ influx should promote voltage hyperpolarisation (just as block of K+ influx causes hyperpolarisation in root hairs28) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity5 to increase Ca2+ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl2, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca2+ ionophore , A2318730 to increase [Ca2+]cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to Vm, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca2+]cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing Vm and [Ca2+]cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.29 While there is still much to do on the “inventory” of PM transporters involved in Ca2+ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action. 相似文献
14.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
15.
Sergey Shabala Lone B?kgaard Lana Shabala Anja T Fuglsang Tracey A Cuin Lev G Nemchinov Michael G Palmgren 《Plant signaling & behavior》2011,6(7):1053-1056
Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper,17 we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.Key words: cytosolic calcium, reactive oxygen species, cross-tolerance, calcium pumpThe phenomenon of cross-tolerance to a variety of biotic and abiotic stresses is well-known.1,2 Some of the demonstrated examples include the correlation between oxidative stress tolerance and pathogen resistance.3–5 At the mechanistic level, changes in cytosolic Ca2+ levels [Ca2+]cyt, have long been implicated as a quintessential component of this process.6 The rise in [Ca2+]cyt is proven to be essential for the development of the oxidative burst required for triggering the activation of several plant defense reactions.7,8 The observed elevation in H2O2 level is believed to result from Ca2+-dependent activation of the NADPH oxidase,8 which then causes a further increase in [Ca2+]cyt via a positive feedback mechanism. This process is further accomplished by defense gene activation, phytoalexin synthesis and eventual cell death.9 Downstream from the stimulus-induced [Ca2+]cyt elevation, cells possess an array of proteins that can respond to a message. Such proteins include calmodulin (CaM),10 Ca2+-dependent protein kinases11 and CaM binding proteins.12 Of note is that when Ca2+ channels are blocked, biosynthesis of ROS is prevented.13While the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the involvement of active Ca2+ efflux systems in this process. In contrast, in animal systems the essential role of re-establishing [Ca2+]cyt to resting levels is widely reported. A sustained increase in [Ca2+]cyt in the alveolar macrophage is thought to be the consequence of membrane Ca2+-ATPase dysfunction.14 In endothelial cells, inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria was named as one of the reasons for [Ca2+]cyt increases.15 A significant loss of the plasma membrane Ca2+-ATPase (PMCA) activity was reported in brain synapses in response to oxidative stress,16 suggesting that PMCA may be a downstream target of oxidative stress.In our recently published paper17 we reported the phenomenon of Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana plants and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. Nonetheless, questions remain, is this transporter the only active Ca2+ efflux system involved in this process?In addition to Ca2+/H+ exchangers, active Ca2+ extrusion could also be achieved by Ca2+-ATPases. Two major types of Ca2+-ATPases that differ substantially in their pharmacology and sensitivity to CaM are known.18 Type P2A pumps (also called ER-type or ECA19,20) are predominantly ER-localized,19 although they are also present at other endomembranes (e.g., tonoplast and Golgi). Four members of this group have been identified in the Arabidopsis genome (named AtECAs 1 to 4).18,21 These pumps lack an N-terminal autoregulatory domain, are insensitive to CaM and suppressed by cyclopropiazonic acid (CPA).19 P2B (or ACA) pumps contain an autoinhibitory N-terminal domain that possesses a binding site for Ca2+-CaM.18 Ten members are known in Arabidopsis (termed AtACA1, 2, 4 and 7 to 13).21 Plant P2B pumps are located at the plasma membrane20 as well as in inner membranes such as tonoplast (e.g., ACA4), ER (e.g., ACA2) and plastids.18,19 These pumps probably constitute the basis for precise cytosolic Ca2+ regulation; as the Ca2+ concentration increases, CaM is activated and binds to the autoinhibitory domain of the Ca2+ pump. This results in the activation of the pump.In our recent study,17 we found no significant difference between the purified plasma membranes fractions isolated from control and UV-treated tobacco plants (with or without PVX inoculation) either in the Ca2+-ATPase activity or in the Ca2+-ATPase expression level and its ability to bind CaM. This suggests that the plasma membrane P2B type pumps (the only pump type known to be expressed at the plasma membrane) play no major role in removing excess Ca2+ from the cytosol under oxidative stress conditions. This led to an obvious question: what about endomembrane Ca2+-ATPases?To address this issue, microsomal membrane fractions were isolated from tobacco leaves in a manner previously described for plasma membrane fractions17 (Fig. 1A). Western blot and CaM overlay assays were then made to investigate the role of endomembrane P2B Ca2+-ATPases in our reported phenomena of acquired resistance. The results show that the expression of the P2B Ca2+ pumps in PVX-inoculated plants is significantly higher than in control plants (Fig. 1B), correlating well with the CaM overlay assay (Fig. 1C). As no difference was observed for the P2B Ca2+-ATPase expression levels in the plasma membranes,17 the observed difference in the microsomal fractions of PVX-infected plants must be due to an increased expression of endomembrane P2B Ca2+-ATPases. Given the fact that Ca2+ pumps have a high affinity for calcium, the observed increase in endomembrane P2B-type Ca2+-ATPases expression in PVX-inoculated plants may be advantageous for more efficient Ca2+ removal from the cytosol into internal organelles.Open in a separate windowFigure 1Expression of P2B Ca2+ in purified microsomal fractions from tobacco leaves. Measurements were undertaken C = mock controls; C-UV = mock controls treated with UV-light; PVX = PVX infected plants; PVX-UV = PVX inoculated plants treated with UV-light. (A) Coomassie Brilliant Blue-stained gel; (B) Protein blot immunostained with a non isoform-specific polyclonal antibody for P2B Ca2+-ATPases; (C) CaM overlay assay.To decipher the possible role of P2A Ca2+-ATPases in acquired resistance, a series of electrophysiological experiments were conducted using inhibitors of P2A-type Ca2+-ATPases, such as thapsigargin (TG)22 and cyclopiazonic acid (CPA).23 Ion-selective Ca2+ microelectrodes were prepared as described elsewhere in reference 24 and 25, and net Ca2+ fluxes were measured from tobacco mesophyll tissue following previously described protocols.17 Leaf pre-treatment for 2 h in either of these inhibitors dramatically suppressed the net Ca2+ efflux measured from tobacco mesophyll cells 2 h after UV light exposure (Fig. 2). Given the specificity of TG and CPA inhibitors for P2A-type Ca2+-ATPases, these results strongly support a hypothesis that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in plant adaptive responses to oxidative stress. This is achieved by removing excess Ca2+ from the cytosol.Open in a separate windowFigure 2Effect of known Ca2+-ATPase blockers on light-induced Ca2+ flux kinetics after 20 min of UV-C treatment. Leaf mesophyll segments were pre-treated in either 5 µM TG (thapsigargin) or 50 µM CPA (cyclopiazonic acid) for 1–1.5 h prior to exposure to UV-C light. Net Ca2+ fluxes were measured 2 h after the end of UV treatment. These were compared with two controls: (1) no pre-treatment/no UV exposure (closed circles) and (2) no pre-treatment/20 min UV exposure (open squares). Mean ± SE (n = 4 to 7).Combining these results with our previously reported observations in reference 17, the following model is proposed (Fig. 3). Oxidative stress (such as UV) causes increased ROS production in leaf chloroplasts, leading to the elevated [Ca2+]cyt. Several Ca2+ efflux systems are involved in restoring basal cytosolic Ca2+ levels. Two of these, the plasma membrane Ca2+/H+ exchanger17 and endomembrane P2A and P2B Ca2+-ATPases (as reported in this study) are upregulated in PVX inoculated plants and contribute to the improved tolerance to oxidative stress. Overall, these findings highlight the potential role of Ca2+ efflux systems in virus-induced tolerance to oxidative stress in plants. This is consistent with our previous reports on the important role of Ca2+ efflux systems in biotic stress tolerance26 and brings forth possibilities for genetic engineering of more tolerant plants by targeting expression and regulation of active Ca2+ efflux systems at either the plasma or endomembranes.Open in a separate windowFigure 3The proposed model of oxidative stress signaling and the role of Ca2+-efflux systems in acquired resistance and plant adaptation to oxidative stress.Overall, a better adaptation of virus-infected plants to a short wave UV irradiation as compared to uninfected controls may suggest that infection triggers common defense mechanisms that could be efficient against secondary unrelated stresses. This observation may lead to the development of novel strategies to protect plants against complex environmental stress conditions. 相似文献
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Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.4 In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.7 Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.8 Terras and his colleagues9 isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.10 A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.11 In addition, over 1,000 DEFL genes have been identified from plant EST projects.12Unlike the insect and mammalian defensins, which are mainly active against bacteria,2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.14 Most plant defensins are involved in defense against a broad range of fungi.2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).2 Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana16 and alter growth of various tomato organs which can assume multiple functions related to defense and development.4 相似文献
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The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization.1,2 The nature of the primary step of its action (alteration in membrane composition,3 transient influx of Ca2+ etc.,2) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling4 but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca2+ and generation of AP series.5 TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.Key words: action potential, cold, liverwort, menthol, thermoreceptors, voltage transient 相似文献