Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

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 isoforms^11–13 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.     

Control of depolarization-evoked presynaptic neurotransmitter release by Cav2.1 calcium channel: old story, new insights

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 Ca_V2.1 and Ca_V2.2 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 Ca_V2.1 and Ca_V2.2 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, Ca_V2.1 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 Ca_V1.2 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 Ca_V2.1 channel.¹² Using the two-electrode voltage-clamp technique on BAPTA-injected Xenopus oocyte expressing the human Ca_V2.1 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, Ca_V2.1 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 Ca_V2.1 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 Ca_V2.1 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 Ca_V1.2 and Ca_V2.2.¹⁵ While expression of Ca_V2.1 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 Ca_V2.1 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), Ca_V2.1 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 Ca_V2.1 channel and vesicle release machinery. At resting membrane potential, Ca_V2.1 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 Ca_V2.1 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 Ca_V2.1 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 Ca_V2.1 channel, as already done for L-type channels (Ca_V1.2).^7,9,10 Moreover, the possibility that Ca_V2.1 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 Ca_V1.2 channels modifies secretion kinetics of catecholamine in chromaffin cells.¹⁶ It is also well known that the auxiliary β-subunit of VGCCs modulates Ca_V2.1 gating modes.¹⁷ Therefore, comparing secretion mediated by a non-conducting Ca_V2.1 channel in the presence of different types of β-subunits would provide important information on the molecular mechanisms through which Ca_V2.1 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.     

Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum,revealed a variability in PME proteolytic maturation

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.     

Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi

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.     

Voltage,reactive oxygen species and the influx of calcium

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 (V_m) 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 V_m, 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 V_m 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 V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 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 V_m 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 DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m 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 flux^14,16 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 V_m and decreases at very depolarized V_m.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 V_m, 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 deficiency^22,23 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 {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ 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 V_m, 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 V_m 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.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

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).     

Endomembrane Ca2+-ATPases play a significant role in virus-induced adaptation to oxidative stress

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 P_2A and P_2B 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 P_2A pumps (also called ER-type or ECA^19,20) 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 P_2B (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 P_2B 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 P_2B 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 P_2B Ca²⁺-ATPases in our reported phenomena of acquired resistance. The results show that the expression of the P_2B 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 P_2B 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 P_2B Ca²⁺-ATPases. Given the fact that Ca²⁺ pumps have a high affinity for calcium, the observed increase in endomembrane P_2B-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 P_2B 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 P_2B Ca²⁺-ATPases; (C) CaM overlay assay.To decipher the possible role of P_2A Ca²⁺-ATPases in acquired resistance, a series of electrophysiological experiments were conducted using inhibitors of P_2A-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 P_2A-type Ca²⁺-ATPases, these results strongly support a hypothesis that both endomembrane P_2A and P_2B 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 P_2A and P_2B 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.     

Plant defensins: Defense,development and application

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     

Electrophysiological approach to examine a putative cold- and menthol-sensitive channel in the liverwort Conocephalum conicum

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