Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.     

Rapid spatiotemporal patterning of cytosolic Ca2+ underlies flagellar excision in Chlamydomonas reinhardtii.

Ca(2+)-dependent signalling processes are implicated in many aspects of flagella function in the green alga, Chlamydomonas. In this study, we examine the spatiotemporal dynamics of cytosolic Ca2+ ([Ca2+](cyt)) in single Chlamydomonas cells during the process of flagellar excision, using biolistically loaded calcium-responsive dyes. Acid-induced deflagellation occurred in parallel with a single transient elevation in whole-cell [Ca2+](cyt), which was absent in the acid deflagellation-deficient adf1 mutant. Deflagellation could also be induced by elevated external Ca2+ ([Ca2+](ext)), which promoted very rapid spiking of [Ca2+](cyt) across the whole cell and in the flagella. We also detected very rapid apically localised Ca2+ signalling events with an approximate duration of 500 msec. Ninety-seven per cent of deflagellation events coincided with a rapid elevation in [Ca2+](cyt) in the apical region of the cell, either in the form of a whole cell or an apically localised increase, indicating that [Ca2+](cyt) elevations in the apical region play an underlying role in deflagellation. Our data indicate that elevated [Ca2+](ext) acts to disrupt Ca2+ homeostasis which induces deflagellation by both Adf1-dependent and Adf1-independent mechanisms. Elevated [Ca2+](ext) also results in further [Ca2+](cyt) elevations after the main period of whole cell spiking which are very strongly associated with deflagellation, exhibit a high degree of apical localisation and are largely absent in the adf1 mutant. We propose that these later elevations may act as specific signals for deflagellation.     

EGF receptor—mediated intracellular calcium increase in human hepatoma BEL—7404 cells

Epidermal growth factor(EGF) induced intracellular free calcium ([Ca^2 ]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line.Single cell[Ca^2 ]i analysis and [Ca^2 ]i measurement in cell populations revealed that EGF triggered a rapid[Ca^2 ]i increase in the dose-dependent and time-dependent manner.Pretreatment of cells with an endoplasmic reticulum(ER) Ca^2 -ATPase inhibitor,thapsigargin(TG) at 100nM concentration for 20 min,completely abolished EGF-induced [Ca^2 ]i increase,and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore,the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca^2 ]i response to EGF.The results suggest that EGF receptor-mediated [Ca^2 ]i increase in the human hepatoma cells is essentially dependent on the Ca^2 storage in ER.     

Stimulation of D-2 Dopamine Receptors Decreases the Evoked In Vitro Release of [3H] Acetylcholine from Rat Neostriatum: Role of K+ and Ca2+

Reportedly, stimulation of D-2 dopamine receptors inhibits the depolarization-induced release of acetylcholine from the neostriatum in a cyclic AMP-independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D-2 receptor-mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D-2 receptor-mediated decrease of K+-evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D-2 stimulation on the K+-evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D-2 receptors in the absence of Ca2+ also inhibits the veratrine-evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D-2 dopamine receptor mediates the decrease of depolarization-induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.     

Internal Ca2+ Mobilization by Muscarinic Stimulation Increases Secretion from Adrenal Chromaffin Cells Only in the Presence of Ca2+ Influx

The cytosolic free Ca2+ concentration ([Ca2+]in) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]in and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]in by activating both Ca2+ influx and intracellular Ca2+ mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]in by mobilizing intracellular Ca2+ but did not cause CA secretion. The MCh-induced rise in [Ca2+]in in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]in elevated above basal values was maintained by a continuous Ca2+ influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]in in a particular region of the cell.     

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.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca2+ ionophore on mitochondria in intact cells

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i). ferutinin, an electrogenic naturally occurring ionophore, and (ii). the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.     

Ca2+在植物盐胁迫响应机制中的调控作用

对植物而言,Ca2+不仅作为一种必须的营养元素,更重要的是作为耦联胞外信号与胞内生理反应的第二信使,当植物受到外界的环境刺激时,细胞中Ca2+会出现变化,引起一系列保护性生理反应,从而减轻环境胁迫对植物体的伤害.我国盐碱地面积广阔,极大地限制了作物种植和农业生产.大量研究表明,Ca2+可以提高植物对盐胁迫的抗性,针对盐胁迫对植物的伤害机制,重点讨论了盐胁迫条件下Ca2+参与的植物体内有关响应途径及作用机制.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.     

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.     

Calcium imaging in Arabidopsis pollen cells using G-CaMP5

Calcium(Ca~(2+)) signaling has been implicated in pollen germination and pollen tube growth. To date,however, we still know very little about how exactly Ca~(2+) signaling links to various physiological subcellular processes during pollen germination and pollen tube growth.Given that Ca~(2+) signaling is tightly related to the cytosolic concentration and dynamics of Ca~(2+), it is vital to trace the dynamic changes in Ca~(2+) levels in order to decode Ca~(2+) signaling. Here, we demonstrate that G-Ca MP5 serves well as an indicator for monitoring cytosolic Ca~(2+) dynamics in pollen cells. Using this probe, we show that cytosolic Ca~(2+) changes dramatically during pollen germination, and, asreported previously, Ca~(2+) forms a tip-focused gradient in the pollen tube and undergoes oscillation in the tip region during pollen tube growth. In particular, using G-CaMP5 allowed us to capture the dynamic changes in the cytosolic Ca~(2+) concentration([Ca~(2+)]_(cyt)) in pollen tubes in response to various exogenous treatments. Our data suggest that G-CaMP5 is a suitable probe for monitoring the dynamics of[Ca~(2+)]_(cyt) in pollen cells.     

The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling

A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5 degrees C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.     

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.