Focus Issue on Calcium Signaling: Identification of Cyclic GMP-Activated Nonselective Ca2+-Permeable Cation Channels and Associated CNGC5 and CNGC6 Genes in Arabidopsis Guard Cells

Cytosolic Ca²⁺ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca²⁺ increases result from Ca²⁺ influx through Ca²⁺-permeable channels in the plasma membrane and Ca²⁺ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca²⁺-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3′,5′-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg²⁺ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba²⁺, Ca²⁺, and Na⁺ ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca²⁺-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO₂ concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca²⁺-permeable cation channels in the plasma membrane of Arabidopsis guard cells.Plants lose water via transpiration and take in CO₂ for photosynthesis through stomatal pores. Each stomatal pore is surrounded by two guard cells, and stomatal movements are driven by the change of turgor pressure in guard cells. The intracellular second messenger Ca²⁺ functions in guard cell signal transduction (Schroeder and Hagiwara, 1989; McAinsh et al., 1990; Webb et al., 1996; Grabov and Blatt, 1998; Allen et al., 1999; MacRobbie, 2000; Mori et al., 2006; Young et al., 2006; Siegel et al., 2009; Chen et al., 2010; Hubbard et al., 2012). Plasma membrane ion channel activity and gene expression in guard cells are finely regulated by the intracellular free calcium concentration ([Ca2+]_cyt; Schroeder and Hagiwara, 1989; Webb et al., 2001; Allen et al., 2002; Siegel et al., 2009; Kim et al., 2010; Stange et al., 2010). Ca²⁺-dependent protein kinases (CPKs) function as targets of the cytosolic Ca²⁺ signal, and several members of the CPK family have been shown to function in stimulus-induced stomatal closing, including the Arabidopsis (Arabidopsis thaliana) CPK3, CPK4, CPK6, CPK10, and CPK11 proteins (Mori et al., 2006; Zhu et al., 2007; Zou et al., 2010; Brandt et al., 2012; Hubbard et al., 2012). Further research found that several CPKs could activate the S-type anion channel SLAC1 in Xenopus laevis oocytes, including CPK21, CPK23, and CPK6 (Geiger et al., 2010; Brandt et al., 2012). At the same time, the Ca²⁺-independent protein kinase Open Stomata1 mediates stomatal closing and activates the S-type anion channel SLAC1 (Mustilli et al., 2002; Yoshida et al., 2002; Geiger et al., 2009; Lee et al., 2009; Xue et al., 2011), indicating that both Ca²⁺-dependent and Ca²⁺-independent pathways function in guard cells.Multiple essential factors of guard cell abscisic acid (ABA) signal transduction function in the regulation of Ca²⁺-permeable channels and [Ca2+]_cyt elevations, including Abscisic Acid Insensitive1 (ABI1), ABI2, Enhanced Response to Abscisic Acid1 (ERA1), the NADPH oxidases AtrbohD and AtrbohF, the Guard Cell Hydrogen Peroxide-Resistant1 (GHR1) receptor kinase, as well as the Ca²⁺-activated CPK6 protein kinase (Pei et al., 1998; Allen et al., 1999, 2002; Kwak et al., 2003; Miao et al., 2006; Mori et al., 2006; Hua et al., 2012). [Ca2+]_cyt increases result from both Ca²⁺ release from intracellular Ca²⁺ stores (McAinsh et al., 1992) and Ca²⁺ influx across the plasma membrane (Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003; Hua et al., 2012). Electrophysiological analyses have characterized nonselective Ca²⁺-permeable channel activity in the plasma membrane of guard cells (Schroeder and Hagiwara, 1990; Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Köhler and Blatt, 2002; Miao et al., 2006; Mori et al., 2006; Suh et al., 2007; Vahisalu et al., 2008; Hua et al., 2012). However, the genetic identities of Ca²⁺-permeable channels in the plasma membrane of guard cells have remained unknown despite over two decades of research on these channel activities.The Arabidopsis genome includes 20 genes encoding cyclic nucleotide-gated channel (CNGC) homologs and 20 genes encoding homologs to animal Glu receptor channels (Lacombe et al., 2001; Kaplan et al., 2007; Ward et al., 2009), which have been proposed to function in plant cells as cation channels (Schuurink et al., 1998; Arazi et al., 1999; Köhler et al., 1999). Recent research has demonstrated functions of specific Glu receptor channels in mediating Ca²⁺ channel activity (Michard et al., 2011; Vincill et al., 2012). Previous studies have shown cAMP activation of nonselective cation currents in guard cells (Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007). However, only a few studies have shown the disappearance of a defined plasma membrane Ca²⁺ channel activity in plants upon mutation of candidate Ca²⁺ channel genes (Ali et al., 2007; Michard et al., 2011; Laohavisit et al., 2012; Vincill et al., 2012). Some CNGCs have been found to be involved in cation nutrient intake, including monovalent cation intake (Guo et al., 2010; Caballero et al., 2012), salt tolerance (Guo et al., 2008; Kugler et al., 2009), programmed cell death and pathogen responses (Clough et al., 2000; Balagué et al., 2003; Urquhart et al., 2007; Abdel-Hamid et al., 2013), thermal sensing (Finka et al., 2012; Gao et al., 2012), and pollen tube growth (Chang et al., 2007; Frietsch et al., 2007; Tunc-Ozdemir et al., 2013a, 2013b). Direct in vivo disappearance of Ca²⁺ channel activity in cngc disruption mutants has been demonstrated in only a few cases thus far (Ali et al., 2007; Gao et al., 2012). In this research, we show that CNGC5 and CNGC6 are required for a cyclic GMP (cGMP)-activated nonselective Ca²⁺-permeable cation channel activity in the plasma membrane of Arabidopsis guard cells.     

A Calcium Sensor-Regulated Protein Kinase,CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, Is Required for Pollen Tube Growth and Polarity

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca²⁺ throughout the bulging tip, whereas LaCl₃, a Ca²⁺ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca²⁺ homeostasis through its potential function in the modulation of Ca²⁺ influx.In flowering plants, fertilization is mediated by pollen tubes that extend directionally toward the ovule for sperm delivery (Krichevsky et al., 2007; Johnson, 2012). The formation of these elongated tubular structures is dependent on extreme polar growth (termed tip growth), in which cell expansion occurs exclusively in the very apical area (Yang, 2008; Rounds and Bezanilla, 2013). As this type of tip growth is amenable to genetic manipulation and cell biological analysis, the pollen tube is an excellent model system for the functional analysis of essential genes involved in polarity control and fertilization (Yang, 2008; Qin and Yang, 2011; Bloch and Yalovsky, 2013).It is well established that Ca²⁺ plays a critical role in pollen germination and tube growth (Konrad et al., 2011; Hepler et al., 2012). A steep tip-focused Ca²⁺ gradient has been detected at the tip of elongating pollen tubes (Rathore et al., 1991; Pierson et al., 1994; Hepler, 1997). In previous studies, artificial dissipation of the Ca²⁺ gradient seriously inhibited tip growth of pollen tubes, whereas elevation of internal Ca²⁺ level induced bending of the growth axis toward the zone of higher Ca²⁺. These studies suggest that Ca²⁺ not only controls pollen tube elongation but also modulates growth orientation (Miller et al., 1992; Malho et al., 1994; Malho and Trewavas, 1996; Hepler, 1997). These Ca²⁺ signatures are perceived and relayed to downstream responses by a complex toolkit of Ca²⁺-binding proteins that function as Ca²⁺ sensors (Yang and Poovaiah, 2003; Harper et al., 2004; Dodd et al., 2010).To date, four major Ca²⁺ sensor families have been identified in Arabidopsis (Arabidopsis thaliana), including calcium-dependent protein kinase, calmodulin (CaM), calmodulin-like (CML), and CALCINEURIN B-LIKE (CBL) proteins (Luan et al., 2002, 2009; Yang and Poovaiah, 2003; Harper et al., 2004). Calcium-dependent protein kinase family members comprise a kinase domain and a CaM-like domain in a single protein; thus, they act not only as a Ca²⁺ sensor but also as an effector, designated as sensor responders (Cheng et al., 2002). In contrast, CaM, CML, and CBL proteins do not have any enzymatic domains but transmit Ca²⁺ signals to downstream targets via Ca²⁺-dependent protein-protein interactions. Therefore, they have been designated as sensor relays (McCormack et al., 2005). While CaM and CML proteins interact with a diverse array of target proteins, it is generally accepted that CBLs interact specifically with a group of Ser/Thr protein kinases termed CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASEs (CIPKs; Luan et al., 2002; Kolukisaoglu et al., 2004).In Arabidopsis, several CBLs coupled with their target CIPKs have been demonstrated to function in the regulation of ion homeostasis and stress responses (Luan et al., 2009). Under salt stress, SALT OVERLY SENSITIVE3 (SOS3)/CBL4-SOS2/CIPK24 regulate SOS1 at the plasma membrane for Na⁺ exclusion, whereas CBL10-CIPK24 complexes appear to regulate Na⁺ sequestration at the tonoplast (Liu et al., 2000; Qiu et al., 2002; Kim et al., 2007; Quan et al., 2007). For low-K⁺ stress, CBL1 and CBL9, with 87% amino acid sequence identity, interact with CIPK23, which regulates a voltage-gated ion channel (ARABIDOPSIS K⁺ TRANSPORTER1) to mediate the uptake of K⁺ in root hairs (Li et al., 2006; Xu et al., 2006; Cheong et al., 2007). In addition, CBL1 integrates plant responses to cold, drought, salinity, and hyperosmotic stresses (Albrecht et al., 2003; Cheong et al., 2003), and CBL9 is involved in abscisic acid signaling and biosynthesis during seed germination (Pandey et al., 2004). Over the past decade, the functions of CBL-CIPK complexes in abiotic stress tolerance have been studied extensively, but only limited studies focus on CBL family members in pollen tube growth. For example, CBL3 overexpression caused a defective phenotype in pollen tube growth (Zhou et al., 2009). Overexpression of CBL1 or its closest homolog CBL9 inhibited pollen germination and perturbed tube growth at high external K⁺, whereas disruption of CBL1 and CBL9 leads to a significantly reduced growth rate of pollen tubes under low-K⁺ conditions (Mähs et al., 2013). The potential roles of CIPKs in pollen tubes so far appear to be completely unknown.In this study, we demonstrated that Arabidopsis CIPK19, a CIPK specifically expressed in pollen grains and pollen tubes, functions in pollen tube tip growth, providing a new insight into the function of the CBL-CIPK network in the control of growth polarity during pollen tube extension in fertilization.     

Involvement of the Sieve Element Cytoskeleton in Electrical Responses to Cold Shocks

This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca²⁺-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca²⁺ influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV₁, reduced by external La³⁺ in keeping with the involvement of Ca²⁺ channels, and a second voltage transient, ΔV₂. Cytochalasin D had no effect on ΔV₁, while ΔV₁ was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca²⁺ influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).It has been argued for a long time that sieve elements (SEs) are devoid of a cytoskeleton (Parthasarathy and Pesacreta, 1980; Thorsch and Esau, 1981; Evert, 1990), but more recent biochemical and cytological studies favor the opposite view. Actin as well as profilin were detected in phloem exudates of various monocot and dicot species (Schobert et al., 1998, 2000), while immunocytochemical tests showed the presence of actin and tubulin in phloem exudates of pumpkin (Cucurbita maxima; Kulikova and Puryaseva, 2002). Proteome analyses gave further credence to the occurrence of microfilaments in SEs in castor bean (Ricinus communis; profilin; Barnes et al., 2004), pumpkin (actin; Walz et al., 2004), canola (Brassica napus; actin, profilin1 and profilin2, actin-depolymerizing factor4; Giavalisco et al., 2006), and rice (Oryza sativa; actin1, actin-depolymerizing factor2, actin depolymerizing-factor3, and actin-depolymerizing factor6; Aki et al., 2008). Moreover, cytological evidence suggests residues of a cytoskeleton in SEs; fluorescent immunolabeling identified an actin/myosin system at the sieve plates (Chaffey and Barlow, 2002).Theoretical considerations also call for the presence of a cytoskeleton in SEs. Turnover and addressing of macromolecules (Fisher et al., 1992; Leineweber et al., 2000) requires a local distribution network in SEs. This function was attributed to an endoplasmic reticulum (ER) continuous to the ER strands running through pore plasmodesma units (Blackman et al., 1998) into the companion cells. Although such a mechanism is essentially conceivable, an interaction between the ER and cytoskeleton would provide a more conventional mode of intracellular distribution (Hepler et al., 1990; Boevink et al., 1998; Ueda et al., 2010; Yokota et al., 2011; Chen et al., 2012). Moreover, macromolecular trafficking through pore plasmodesma units (Lucas et al., 2001) was proposed to be executed by actin and myosin (Oparka, 2004), implying the presence of a cytoskeleton in SEs. Despite the massive circumstantial evidence, however, a complete cytoskeleton network and its spatial distribution in SEs have not been visually documented thus far.The existence of an SE cytoskeleton would raise questions regarding its task(s) in this highly specialized cell type. In other plant cells, the cytoskeleton was proposed to be engaged, among others, in ion channel operation and intracellular signaling (Trewavas and Malho, 1997; Mazars et al., 1997, and refs. therein; Thuleau et al., 1998; Örvar et al., 2000; Sangwan et al., 2001; Drøbak et al., 2004; Davies and Stankovic, 2006), as in animal cells (Janmey, 1998; Lange and Gartzke, 2006). For instance, K⁺ fluxes are regulated by actin dynamics (Hwang et al., 1997; Liu and Luan, 1998; Chérel, 2004), while Ca²⁺ influx into the cytoplasm appears to be mediated by voltage-dependent Ca²⁺-permeable channels associated with microtubules (Mazars et al., 1997; Thion et al., 1998) or by mechanosensitive channels possibly associated with microfilaments (Wang et al., 2004; Zhang et al., 2007).Both types of Ca²⁺-permeable channels probably reside in the SE plasma membrane (Knoblauch et al., 2001; Hafke et al., 2007, 2009; Furch et al., 2009), where they are likely involved in Ca²⁺-dependent systemic signaling (Furch et al., 2009; Hafke et al., 2009; van Bel et al., 2011; Hafke and van Bel, 2013). These channels are also putative initiators of Ca²⁺-induced signal transduction in SEs, leading to sieve-plate occlusion in response to local cold shocks (Thorpe et al., 2010). In fava bean (Vicia faba), Ca²⁺-dependent sieve tube occlusion by dispersion of special phloem-specific proteins (P-proteins) known as forisomes has been studied intensely (Knoblauch et al., 2001; Furch et al., 2007, 2009; Thorpe et al., 2010). Thus, apart from its distributive tasks, a cytoskeleton may be of major importance for intracellular signaling cascades in the highly specialized, sparsely equipped SEs.Our objective was to investigate the existence and spatial distribution of an SE cytoskeleton and its engagement in local signaling through Ca²⁺ influx brought about by cold shocks. This study dealt with the visualization of cytoskeletal components in intact sieve tubes using microinjection of fluorescent phalloidin and immunocytochemistry. Confocal laser-scanning micrography (CLSM) and transmission electron microscopy unequivocally showed a parietally located cylindrical actin meshwork. We demonstrated the engagement of the network in local cold shock-induced electrical responses and its association with Ca²⁺ influx, since we found effects of the Ca²⁺ channel blocker La³⁺ and of the cytoskeleton disruptor latrunculin A (LatA) on electrical signatures triggered by cold shocks and, by consequence, on forisome conformation changes.     

Focus Issue on Calcium Signaling: Calcium and Reactive Oxygen Species Rule the Waves of Signaling

Calcium signaling and reactive oxygen species signaling are directly connected, and both contribute to cell-to-cell signal propagation in plants.Calcium (Ca²⁺) is an important second messenger with diverse functions not only in mammals but also in plants. It is released in response to a variety of stimuli like biotic and abiotic stresses and facilitates a tight regulation of response reactions as well as of developmental processes (Sanders et al., 2002; Steinhorst and Kudla, 2012). Ca²⁺ accumulation events are characterized by distinct temporal and spatial features, and they can vary in terms of amplitude, frequency, and duration (Webb et al., 1996; Scrase-Field and Knight, 2003; Dodd et al., 2010; Kudla et al., 2010). Spatially defined Ca²⁺ signals can be generated due to the especially slow diffusion rate of the Ca²⁺ ion in the cytoplasm in combination with tightly regulated release and uptake from and into different intracellular stores and the apoplast. Together, these characteristics encode information about particular stimuli, for example, drought stress that is presented to the cell as so-called Ca²⁺ signatures (Webb et al., 1996). This information has to be decoded and transmitted by a signaling machinery in order to initiate adequate response reactions, for example, stomatal closure (Allen et al., 2000, 2001; Sanders et al., 2002). Ca²⁺ signatures can be sensed by proteins that bind Ca²⁺ via helix-loop-helix EF-hand motifs. Arabidopsis (Arabidopsis thaliana) possesses at least 250 putative EF-hand proteins, 100 of which have been classified as Ca²⁺ sensor proteins (Day et al., 2002; Hashimoto and Kudla, 2011). Given that each member of this intricate set of Ca²⁺ sensor proteins can exhibit characteristic expression and subcellular localization profiles as well as distinct Ca²⁺ affinities, plants are equipped with a complex signal-decoding machinery to process a wide range of different Ca²⁺ signals (Batistič and Kudla, 2004; Batistič and Kudla, 2010). Ca²⁺ functions in concert with other important second messengers like reactive oxygen species (ROS). ROS can be generated in a controlled manner by several types of enzymes, such as NADPH oxidases, in order to contribute to pathogen defense and cell signaling. Recent findings point to direct connections between ROS and Ca²⁺ signaling pathways that enable cell-to-cell communication and thereby long-distance transmission of signals in plants. In this Update, we focus on new findings in the field of plant Ca²⁺ signaling during the past 3 years since the status of the field was discussed in comprehensive reviews (Dodd et al., 2010; Kudla et al., 2010; Mazars et al., 2011; Reddy et al., 2011) and put special emphasis on the contribution of a plant-specific Ca²⁺ signaling network to deciphering defined Ca²⁺ signals and its integration with ROS signaling.     

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.In vertebrates, ionotropic glutamate receptors (iGluRs in animals) are ligand-gated cation channels that mediate the majority of the excitatory neurotransmission in the central nervous system (Dingledine et al., 1999). In the model plant Arabidopsis (Arabidopsis thaliana), 20 genes encoding homologs of animal iGluRs have been identified (Lam et al., 1998). According to phylogenetic analyses, the Arabidopsis GLUTAMATE RECEPTOR (AtGLR) homologs can be subdivided into three separate subgroups (Chiu et al., 1999, 2002). Some evidence for the channel-forming ability by plant ionotropic glutamate receptors (iGLRs) has been obtained only recently, and only for AtGLR3.4 and AtGLR1.4 expressed in heterologous systems (Vincill et al., 2012; Tapken et al., 2013). Studies with transgenic plants suggested roles of members of the plant GLR family in Ca²⁺ fluxes (AtGLR2; Kim et al., 2001), coordination of mitotic activity in the root apical meristem (Li et al., 2006), regulation of abscisic acid biosynthesis and water balance (AtGLR1.1; Kang and Turano, 2003; Kang et al., 2004), carbon/nitrogen sensing (AtGLR1.1; Kang and Turano, 2003), resistance against fungal infection (Kang et al., 2006), leaf-to-leaf wound signaling (Mousavi et al., 2013), and lateral root initiation (Vincill et al., 2013). Application of antagonists and agonists of animal iGluRs revealed that plant GLRs might be involved in the regulation of root growth and branching (Walch-Liu et al., 2006), in light signal transduction (Lam et al., 1998), and in the response to aluminum (Sivaguru et al., 2003). In various plant cell types, the agonists Glu- and Gly-induced plasma membrane depolarization and a rise in intracellular Ca²⁺ concentration that were inhibited by blockers of nonselective cation channels (NSCCs) and by antagonists of animal iGluRs (Dennison and Spalding, 2000; Dubos et al., 2003; Meyerhoff et al., 2005; Krol et al., 2007; Kwaaitaal et al., 2011; Michard et al., 2011). Furthermore, Glu-activated cation currents in patch-clamped root protoplasts were inhibited by NSCC blockers such as La³⁺ and Gd³⁺ (Demidchik et al., 2004). Therefore, it was proposed that plant iGLRs can form Ca²⁺-permeable NSCCs, are inhibited by animal iGluR antagonists, and might contribute to the shaping of plant Ca²⁺ signaling (McAinsh and Pittman, 2009). Studies using AtGLR3.3 mutant plants showed that intracellular Ca²⁺ rise and membrane depolarization induced by Glu in Arabidopsis hypocotyls and root cells are correlated with the presence of AtGLR3.3 (Qi et al., 2006; Stephens et al., 2008).However, most plant iGLRs, when expressed in heterologous systems, do not give rise to any current (e.g. in Xenopus spp. oocytes) or are toxic to host cells (e.g. in mammalian cells; Davenport, 2002). Recently, to examine whether AtGLR homologs possess functional ion channel domains, Tapken and Hollmann (2008) transplanted the pore loop together with two adjacent intracellular loops of 17 AtGLR subunits into two rat iGluR subunits and tested the resulting chimeric receptors for ion channel activity in the heterologous expression system Xenopus spp. oocyte. They showed that AtGLR1.1 and AtGLR1.4 have functional ion pore domains. The AtGLR1.1 pores are permeable to Na⁺, K⁺, and Ca²⁺ and are blocked by the nonspecific cation channel blocker La³⁺ (Tapken and Hollmann, 2008). Recent work has demonstrated that the expression of full-length AtGLR1.4 in oocytes gives rise to an amino acid-activated, nonselective, calcium-permeable channel that was found to be inhibited by the animal iGluR modulators 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione (Tapken et al., 2013).The study of these channels has so far been restricted to those members that are located in the plasma membrane and were proved to be functional in the expression systems used. Instead, various localization prediction tools suggest that some of the plant GLRs might have chloroplast and mitochondrial targeting. In general, determining the subcellular localization of a protein is an important step toward understanding its function. We recently reported the localization of GLR3.4 to the inner chloroplast membrane (Teardo et al., 2011), which was also shown to harbor a 6,7-dinitroquinoxaline-2,3-dione-sensitive, calcium-permeable channel activity (Teardo et al., 2010). No other studies have addressed the eventual subcellular localization of other putative Glu receptors.In this work, we show that an isoform of GLR3.5 is efficiently targeted to the mitochondria. Functional expression of the channel in this organelle is indicated by the fact that its absence in knockout plants leads to a dramatically altered ultrastructure of mitochondria that impacts the plant physiology, ultimately leading to an anticipated senescence.     

Focus Issue on Calcium Signaling: Calcium-Dependent Protein Kinase CPK6 Positively Functions in Induction by Yeast Elicitor of Stomatal Closure and Inhibition by Yeast Elicitor of Light-Induced Stomatal Opening in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca²⁺-dependent signaling pathway. A Ca²⁺-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca²⁺-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca²⁺ concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K⁺ channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.Stomata, formed by pairs of guard cells, play a critical role in regulation of plant CO₂ uptake and water loss, thus critically influencing plant growth and water stress responsiveness. Guard cells respond to a variety of abiotic and biotic stimuli, such as light, drought, and pathogen attack (Israelsson et al., 2006; Shimazaki et al., 2007; Melotto et al., 2008).Elicitors derived from microbial surface mimic pathogen attack and induce stomatal closure in various plant species such as Solanum lycopersicum (Lee et al., 1999), Commelina communis (Lee et al., 1999), Hordeum vulgare (Koers et al., 2011), and Arabidopsis (Arabidopsis thaliana; Melotto et al., 2006; Khokon et al., 2010). Yeast elicitor (YEL) induces stomatal closure in Arabidopsis (Klüsener et al., 2002; Khokon et al., 2010; Salam et al., 2013). Our recent studies showed that YEL inhibits light-induced stomatal opening and that protein phosphorylation is involved in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening (Salam et al., 2013).Cytosolic Ca²⁺ has long been recognized as a conserved second messenger in stomatal movement (Shimazaki et al., 2007; Roelfsema and Hedrich 2010; Hubbard et al., 2012). Elevation of cytosolic free Ca²⁺ concentration ([Ca2+]_cyt) is triggered by influx of Ca²⁺ from apoplast and release of Ca²⁺ from intracellular stores in guard cell signaling (Leckie et al., 1998; Hamilton et al., 2000; Pei et al., 2000; Garcia-Mata et al., 2003; Lemtiri-Chlieh et al., 2003). The influx of Ca²⁺ is carried by nonselective Ca²⁺-permeable cation (I_Ca) channels that are activated by plasma membrane hyperpolarization and H₂O₂ (Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003). Elevation of [Ca2+]_cyt activates slow anion (S-type) channels and down-regulates inward-rectifying potassium (K_in) channels in guard cells (Schroeder and Hagiwara, 1989; Grabov and Blatt, 1999). The activation of S-type channels is a hallmark of stomatal closure, and the suppression of K_in channels is favorable to stomatal closure but not to stomatal opening (Pei et al., 1997; Kwak et al., 2001; Xue et al., 2011; Uraji et al., 2012).YEL induces stomatal closure with extracellular H₂O₂ production, intracellular H₂O₂ accumulation, activation of I_Ca channels, and transient [Ca2+]_cyt elevations (Klüsener et al., 2002; Khokon et al., 2010). However, it remains to be clarified whether YEL activates S-type channels and inhibits K_in channels in guard cells.Calcium-dependent protein kinases (CDPKs) are regulators in Ca²⁺-dependent guard cell signaling (Mori et al., 2006; Zhu et al., 2007; Geiger et al., 2010, 2011; Zou et al., 2010; Munemasa et al., 2011; Brandt et al., 2012; Scherzer et al., 2012). In guard cells, CDPKs regulate activation of S-type and I_Ca channels and inhibition of K_in channels (Mori et al., 2006; Zou et al., 2010; Munemasa et al., 2011). A CDPK, CPK6, positively regulates activation of S-type channels and I_Ca channels without affecting H₂O₂ production in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure (Mori et al., 2006; Munemasa et al., 2011). CPK6 phosphorylates and activates SLOW ANION CHANNEL-ASSOCIATED1 expressed in Xenopus spp. oocyte (Brandt et al., 2012; Scherzer et al., 2012). These findings underline the role of CPK6 in regulation of ion channel activation and stomatal movement, leading us to test whether CPK6 regulates the induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening.In this study, we investigated activation of S-type channels and inhibition of K_in channels by YEL and roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. For this purpose, we examined the effects of mutation of CPK6 on induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening, activation of I_Ca channels, transient [Ca2+]_cyt elevations, activation of S-type channels, inhibition of K_in channels, H₂O₂ production in leaves, and H₂O₂ accumulation in leaves and guard cells.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Focus Issue on Calcium Signaling: Calcium-Dependent and -Independent Stomatal Signaling Network and Compensatory Feedback Control of Stomatal Opening via Ca2+ Sensitivity Priming

Guard cells use compensatory feedback controls to adapt to conditions that produce excessively open stomata.In the past 15 years or more, many mutants that are impaired in stimulus-induced stomatal closing and opening have been identified and functionally characterized in Arabidopsis (Arabidopsis thaliana), leading to a mechanistic understanding of the guard cell signal transduction network. However, evidence has only recently emerged that mutations impairing stomatal closure, in particular those in slow anion channel SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), unexpectedly also exhibit slowed stomatal opening responses. Results suggest that this compensatory slowing of stomatal opening can be attributed to a calcium-dependent posttranslational down-regulation of stomatal opening mechanisms, including down-regulation of inward K⁺ channel activity. Here, we discuss this newly emerging stomatal compensatory feedback control model mediated via constitutive enhancement (priming) of intracellular Ca²⁺ sensitivity of ion channel activity. The CALCIUM-DEPENDENT PROTEIN KINASE6 (CPK6) is strongly activated by physiological Ca²⁺ elevations and a model is discussed and open questions are raised for cross talk among Ca²⁺-dependent and Ca²⁺-independent guard cell signal transduction pathways and Ca²⁺ sensitivity priming mechanisms.Stomatal pores formed by two guard cells enable CO₂ uptake from the atmosphere, but also ensure leaf cooling and provide a pulling force for nutrient uptake from the soil via transpiration. These vitally important processes are inevitably accompanied by water loss through stomata. Stomatal opening and closure is caused by the uptake and release of osmotically active substances and is tightly regulated by signaling pathways that lead to the activation or inactivation of guard cell ion channels and pumps. Potassium ions enter guard cells through the inward-rectifying K⁺ channels (K+_in) during stomatal opening and are released via outward-rectifying K⁺ channels during stomatal closure (Schroeder et al., 1987; Hosy et al., 2003; Roelfsema and Hedrich 2005). Cytosolic Ca²⁺, an important second messenger in plants, mediates ion channel regulation, particularly down-regulation of inward-conducting K+_in channels and activation of S-type anion channels, thus mediating stomatal closure and inhibiting stomatal opening (Schroeder and Hagiwara, 1989; Dodd et al., 2010; Kim et al., 2010). Stomatal closure is initiated by anion efflux via the slow S-type anion channel SLAC1 (Negi et al., 2008; Vahisalu et al., 2008; Kollist et al., 2011) and the voltage-dependent rapid R-type anion channel QUICK-ACTIVATING ANION CHANNEL1 (Meyer et al. 2010; Sasaki et al., 2010).In recent years, advances have been made toward understanding mechanisms mediating abscisic acid (ABA)-induced stomatal closure (Cutler et al., 2010; Kim et al., 2010; Raghavendra et al., 2010). The core ABA signaling module, consisting of PYR/RCAR (for pyrabactin resistance 1/regulatory components of ABA receptors) receptors, clade A protein phosphatases (PP2Cs), SNF-related protein kinase OPEN STOMATA1 (OST1), and downstream targets, is Ca²⁺-independent (Ma et al., 2009; Park et al., 2009; Hubbard et al., 2010). However, ABA-induced stomatal closure was reduced to only 30% of the normal stomatal closure response under conditions that inhibited intracellular cytosolic free calcium ([Ca2+]_cyt) elevations in Arabidopsis (Siegel et al., 2009), consistent with previous findings in other plants (De Silva et al., 1985; Schwartz, 1985; McAinsh et al., 1991; MacRobbie, 2000). Together these and other studies show the importance of [Ca2+]_cyt for a robust ABA-induced stomatal closure. Here, we discuss Ca²⁺-dependent and Ca²⁺-independent signaling pathways in guard cells and open questions on how these may work together.Plants carrying mutations in the SLAC1 anion channel have innately more open stomata, and exhibit clear impairments in ABA-, elevated CO₂-, Ca²⁺-, ozone-, air humidity-, darkness-, and hydrogen peroxide-induced stomatal closure (Negi et al., 2008; Vahisalu et al., 2008; Merilo et al., 2013). Recent research, however, unexpectedly revealed that mutations in SLAC1 also down-regulate stomatal opening mechanisms and slow down stomatal opening (Laanemets et al., 2013).