Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells

Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.     

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Copper elicits an increase in cytosolic free calcium in cultured tobacco cells

At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.     

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

Direct cytoplasmic CA(2+) responses to gastrin-releasing peptide in single beta cells

The neuropeptide gastrin releasing peptide (GRP) stimulates insulin secretion and induces oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) in clonal insulinoma cells. It is not known whether GRP affects [Ca(2+)](cyt) in normal beta cells. We investigated, in single, normal, mouse islet beta cells, the effects of GRP on [Ca(2+)](cyt), by dual wavelength spectrophotofluorometry. Beta cells were identified by their typical response to glucose or tolbutamide. At 15 mM glucose, GRP (100 nM) evoked an immediate [Ca(2+)](cyt) transient to 423 +/- 48 nM compared to 126 +/- 18 nM before GRP (P < 0.001). After the initial transient, [Ca(2+)](cyt) exhibited either a sustained elevation or oscillations. At 3.3 mM glucose, in cells with a non-oscillating [Ca(2+)](cyt), GRP stimulated a prompt increase in [Ca(2+)](cyt) (from 60 +/- 6 to 285 +/- 30 nM, P = 0.024) followed by either a sustained increase in [Ca(2+)](cyt) or [Ca(2+)](cyt) oscillations. We conclude that GRP promptly elevates [Ca(2+)](cyt) by a direct action in normal mouse pancreatic beta cells.     

Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice

Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca(2+)-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca(2+)-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Sugar-induced increase in cytosolic Ca(2+) in Arabidopsis thaliana whole plants.

Using Ca(2+)-dependent photoprotein aequorin-transformed Arabidopsis thaliana, sugar-induced increase in cytosolic free Ca(2+ )concentration ([Ca(2+)](cyt))( )was investigated by luminescence imaging technique. When 0.1 M sucrose was fed to roots of autotrophically grown intact whole plants whose roots had been incubated overnight with coelenterazine to reconstitute aequorin systemically, strong and transient (within 20 s) luminescence was observed in the roots; that luminescence was followed by weak luminescence moving from the lower leaves to the upper leaves. The moving rate of luminescence was roughly comparable to that of [(14)C]sucrose. Application of 0.1 M glucose or fructose induced transient luminescence in excised leaves. No such luminescence was observed in heterotrophically grown (with sucrose) whole plants or in excised tissues. mRNA levels of sucrose-H(+) symporter genes AtSUC1 and AtSUC2 were higher in autotrophic plants than in heterotrophic plants. These results indicate that influx of transported sucrose together with H(+) into the mesophyll cells of autotrophic plants may depolarize the membrane potential, and subsequently activate a voltage-gated Ca(2+) channel on the plasma membrane, resulting in a [Ca(2+)](cyt) increase. The [Ca(2+)](cyt) increase might initiate Ca(2+ )signaling leading to the expression of genes related to biosynthesis of storage carbohydrates. Hexoses, when applied, might also be involved in the [Ca(2+)](cyt) increase mediated by monosaccharide-H(+) co-transporters.     

An osmotically induced cytosolic Ca2+ transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance of Saccharomyces cerevisiae

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration ( approximately 1 min) transient cytosolic Ca(2+) ([Ca(2+)](cyt)) increase (Ca(2+)-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca(2+)](cyt) transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca(2+) transport, or mutations (cch1Delta or mid1Delta) that reduce Ca(2+) influx, indicating that Ca(ext)(2+) is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 m) or sorbitol (0.8 or 1.0 m) enhanced the subsequent growth of these cells in media containing 1 m NaCl or 2 m sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1Delta mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mm CaCl(2). Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca(2+)](cyt) transient that is facilitated by Ca(2+) influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1Delta mutation, and FK506, indicating that the [Ca(2+)](cyt) transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.     

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.