Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca²⁺ channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺-resistant and an Al³⁺-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca²⁺) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca²⁺ in the cytoplasm ([Ca²⁺]_cyt) in response to external and intracellular signals. These changes in [Ca²⁺]_cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca²⁺]_cyt fluctuations occur because cells have a Ca²⁺ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca²⁺-binding proteins. The on switches depend on membrane-localized Ca²⁺ channels that control the entry of Ca²⁺ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers in the plasma membrane or endomembrane that remove Ca²⁺ from the cytosol, bringing the [Ca²⁺]_cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca²⁺ have led investigators to ask how specificity in Ca²⁺ signaling is maintained. It has been proposed that specificity in Ca²⁺ signaling is achieved because a particular stimulus elicits a distinct Ca²⁺ signature, which is defined by the timing, magnitude, and frequency of [Ca²⁺]_cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca²⁺]_cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca²⁺ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca²⁺]_cyt response (Monshausen et al., 2009). Translating the Ca²⁺ signature into a defined cellular response is governed by a number of Ca²⁺-binding proteins such as calreticulin that act as [Ca²⁺]_cyt buffers, which shape both the amplitude and duration of the Ca²⁺ signal or Ca²⁺ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca²⁺ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca²⁺]_cyt in the cell. Such measurements have been conducted using Ca²⁺-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca²⁺ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca²⁺]_cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca²⁺ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca²⁺]_cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca²⁺]_cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca²⁺]_cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca²⁺]_cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca²⁺]_cyt changes in Arabidopsis seedling roots after aluminum (Al³⁺) exposure. Although Ca²⁺ signaling has long been implicated in mediating Al³⁺ responses in plants (Rengel and Zhang, 2003), the [Ca²⁺]_cyt changes evoked by Al³⁺ reported in the literature have been inconsistent, and as such, the significance of these [Ca²⁺]_cyt responses to mechanisms of Al³⁺ toxicity are not very clear. For instance, some studies reported that Al³⁺ caused a decrease in [Ca²⁺]_cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca²⁺]_cyt upon Al³⁺ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca²⁺]_cyt within seconds of Al³⁺ exposure. The general pattern of [Ca²⁺]_cyt changes observed after Al³⁺ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca²⁺]_cyt signaling. We also observed root zone-dependent variations in the [Ca²⁺]_cyt signatures evoked by Al³⁺ in regard to the shape, duration, and timing of the [Ca²⁺]_cyt response. Other trivalent ions such as lanthanum (La³⁺) and gadolinium (Ga³⁺), which have been widely used as Ca²⁺ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca²⁺]_cyt in root cells that were similar to those elicited by Al³⁺. Al³⁺, La³⁺, and Gd³⁺ elicited similar [Ca²⁺]_cyt signatures in the Al³⁺-tolerant mutant alr104 (Larsen et al., 1998) and the Al³⁺-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca²⁺]_cyt increases we report here may not be tightly linked to mechanisms of Al³⁺ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca²⁺]_cyt signatures in different root developmental zones that should be useful for future studies on Ca²⁺ signaling mechanisms in plants.     

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

Although the role of Ca²⁺ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca²⁺ efflux systems in this process. In our recent paper,¹⁷ we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca²⁺/H⁺ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P_2A and P_2B Ca²⁺-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca²⁺ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca²⁺ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.Key words: cytosolic calcium, reactive oxygen species, cross-tolerance, calcium pumpThe phenomenon of cross-tolerance to a variety of biotic and abiotic stresses is well-known.^1,2 Some of the demonstrated examples include the correlation between oxidative stress tolerance and pathogen resistance.^3–5 At the mechanistic level, changes in cytosolic Ca²⁺ levels [Ca²⁺]_cyt, have long been implicated as a quintessential component of this process.⁶ The rise in [Ca²⁺]_cyt is proven to be essential for the development of the oxidative burst required for triggering the activation of several plant defense reactions.^7,8 The observed elevation in H₂O₂ level is believed to result from Ca²⁺-dependent activation of the NADPH oxidase,⁸ which then causes a further increase in [Ca²⁺]_cyt via a positive feedback mechanism. This process is further accomplished by defense gene activation, phytoalexin synthesis and eventual cell death.⁹ Downstream from the stimulus-induced [Ca²⁺]_cyt elevation, cells possess an array of proteins that can respond to a message. Such proteins include calmodulin (CaM),¹⁰ Ca²⁺-dependent protein kinases¹¹ and CaM binding proteins.¹² Of note is that when Ca²⁺ channels are blocked, biosynthesis of ROS is prevented.¹³While the role of Ca²⁺ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the involvement of active Ca²⁺ efflux systems in this process. In contrast, in animal systems the essential role of re-establishing [Ca²⁺]_cyt to resting levels is widely reported. A sustained increase in [Ca²⁺]_cyt in the alveolar macrophage is thought to be the consequence of membrane Ca²⁺-ATPase dysfunction.¹⁴ In endothelial cells, inhibition of the Ca²⁺/Na⁺ electroneutral exchanger of the mitochondria was named as one of the reasons for [Ca²⁺]_cyt increases.¹⁵ A significant loss of the plasma membrane Ca²⁺-ATPase (PMCA) activity was reported in brain synapses in response to oxidative stress,¹⁶ suggesting that PMCA may be a downstream target of oxidative stress.In our recently published paper¹⁷ we reported the phenomenon of Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana plants and showed the critical role of plasma membrane Ca²⁺/H⁺ exchangers in this process. Nonetheless, questions remain, is this transporter the only active Ca²⁺ efflux system involved in this process?In addition to Ca²⁺/H⁺ exchangers, active Ca²⁺ extrusion could also be achieved by Ca²⁺-ATPases. Two major types of Ca²⁺-ATPases that differ substantially in their pharmacology and sensitivity to CaM are known.¹⁸ Type P_2A pumps (also called ER-type or ECA^19,20) are predominantly ER-localized,¹⁹ although they are also present at other endomembranes (e.g., tonoplast and Golgi). Four members of this group have been identified in the Arabidopsis genome (named AtECAs 1 to 4).^18,21 These pumps lack an N-terminal autoregulatory domain, are insensitive to CaM and suppressed by cyclopropiazonic acid (CPA).¹⁹ P_2B (or ACA) pumps contain an autoinhibitory N-terminal domain that possesses a binding site for Ca²⁺-CaM.¹⁸ Ten members are known in Arabidopsis (termed AtACA1, 2, 4 and 7 to 13).²¹ Plant P_2B pumps are located at the plasma membrane²⁰ as well as in inner membranes such as tonoplast (e.g., ACA4), ER (e.g., ACA2) and plastids.^18,19 These pumps probably constitute the basis for precise cytosolic Ca²⁺ regulation; as the Ca²⁺ concentration increases, CaM is activated and binds to the autoinhibitory domain of the Ca²⁺ pump. This results in the activation of the pump.In our recent study,¹⁷ we found no significant difference between the purified plasma membranes fractions isolated from control and UV-treated tobacco plants (with or without PVX inoculation) either in the Ca²⁺-ATPase activity or in the Ca²⁺-ATPase expression level and its ability to bind CaM. This suggests that the plasma membrane P_2B type pumps (the only pump type known to be expressed at the plasma membrane) play no major role in removing excess Ca²⁺ from the cytosol under oxidative stress conditions. This led to an obvious question: what about endomembrane Ca²⁺-ATPases?To address this issue, microsomal membrane fractions were isolated from tobacco leaves in a manner previously described for plasma membrane fractions¹⁷ (Fig. 1A). Western blot and CaM overlay assays were then made to investigate the role of endomembrane P_2B Ca²⁺-ATPases in our reported phenomena of acquired resistance. The results show that the expression of the P_2B Ca²⁺ pumps in PVX-inoculated plants is significantly higher than in control plants (Fig. 1B), correlating well with the CaM overlay assay (Fig. 1C). As no difference was observed for the P_2B Ca²⁺-ATPase expression levels in the plasma membranes,¹⁷ the observed difference in the microsomal fractions of PVX-infected plants must be due to an increased expression of endomembrane P_2B Ca²⁺-ATPases. Given the fact that Ca²⁺ pumps have a high affinity for calcium, the observed increase in endomembrane P_2B-type Ca²⁺-ATPases expression in PVX-inoculated plants may be advantageous for more efficient Ca²⁺ removal from the cytosol into internal organelles.Open in a separate windowFigure 1Expression of P_2B Ca²⁺ in purified microsomal fractions from tobacco leaves. Measurements were undertaken C = mock controls; C-UV = mock controls treated with UV-light; PVX = PVX infected plants; PVX-UV = PVX inoculated plants treated with UV-light. (A) Coomassie Brilliant Blue-stained gel; (B) Protein blot immunostained with a non isoform-specific polyclonal antibody for P_2B Ca²⁺-ATPases; (C) CaM overlay assay.To decipher the possible role of P_2A Ca²⁺-ATPases in acquired resistance, a series of electrophysiological experiments were conducted using inhibitors of P_2A-type Ca²⁺-ATPases, such as thapsigargin (TG)²² and cyclopiazonic acid (CPA).²³ Ion-selective Ca²⁺ microelectrodes were prepared as described elsewhere in reference ²⁴ and ²⁵, and net Ca²⁺ fluxes were measured from tobacco mesophyll tissue following previously described protocols.¹⁷ Leaf pre-treatment for 2 h in either of these inhibitors dramatically suppressed the net Ca²⁺ efflux measured from tobacco mesophyll cells 2 h after UV light exposure (Fig. 2). Given the specificity of TG and CPA inhibitors for P_2A-type Ca²⁺-ATPases, these results strongly support a hypothesis that both endomembrane P_2A and P_2B Ca²⁺-ATPases play significant roles in plant adaptive responses to oxidative stress. This is achieved by removing excess Ca²⁺ from the cytosol.Open in a separate windowFigure 2Effect of known Ca²⁺-ATPase blockers on light-induced Ca²⁺ flux kinetics after 20 min of UV-C treatment. Leaf mesophyll segments were pre-treated in either 5 µM TG (thapsigargin) or 50 µM CPA (cyclopiazonic acid) for 1–1.5 h prior to exposure to UV-C light. Net Ca²⁺ fluxes were measured 2 h after the end of UV treatment. These were compared with two controls: (1) no pre-treatment/no UV exposure (closed circles) and (2) no pre-treatment/20 min UV exposure (open squares). Mean ± SE (n = 4 to 7).Combining these results with our previously reported observations in reference ¹⁷, the following model is proposed (Fig. 3). Oxidative stress (such as UV) causes increased ROS production in leaf chloroplasts, leading to the elevated [Ca²⁺]_cyt. Several Ca²⁺ efflux systems are involved in restoring basal cytosolic Ca²⁺ levels. Two of these, the plasma membrane Ca²⁺/H⁺ exchanger¹⁷ and endomembrane P_2A and P_2B Ca²⁺-ATPases (as reported in this study) are upregulated in PVX inoculated plants and contribute to the improved tolerance to oxidative stress. Overall, these findings highlight the potential role of Ca²⁺ efflux systems in virus-induced tolerance to oxidative stress in plants. This is consistent with our previous reports on the important role of Ca²⁺ efflux systems in biotic stress tolerance²⁶ and brings forth possibilities for genetic engineering of more tolerant plants by targeting expression and regulation of active Ca²⁺ efflux systems at either the plasma or endomembranes.Open in a separate windowFigure 3The proposed model of oxidative stress signaling and the role of Ca²⁺-efflux systems in acquired resistance and plant adaptation to oxidative stress.Overall, a better adaptation of virus-infected plants to a short wave UV irradiation as compared to uninfected controls may suggest that infection triggers common defense mechanisms that could be efficient against secondary unrelated stresses. This observation may lead to the development of novel strategies to protect plants against complex environmental stress conditions.     

Voltage,reactive oxygen species and the influx of calcium

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca²⁺ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca²⁺ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca²⁺ influx, suggesting that these enzymes are involved in sensing Ca²⁺ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) encodes plant cell signals.¹ Reactive oxygen species (ROS) are potent regulators of the PM Ca²⁺ channels implicated in signalling and developmental increases in [Ca²⁺]_cyt.^1,2 Plasma membrane (PM) voltage (V_m) also plays a significant part in generating specific [Ca²⁺]_cyt elevations through the opening of voltage-gated Ca²⁺-permeable channels, allowing Ca²⁺ influx.^1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca²⁺ channels (HACCs),⁴ ROS-activated HACCs⁵ and depolarisation-activated Ca²⁺ channels (DACCs).⁶ The DACC characterisation pointed to the presence of a Cl⁻-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.⁶ This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.⁷ Previous studies have shown that root hair PM also harbors K⁺ channels (mediating inward or outward flux)^8–10 and a H⁺-ATPase.¹¹ A key problem to address now is how these transporters interact to generate and be influenced by PM V_m, thus gating and in turn being regulated by their companion Ca²⁺ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between V_m and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.¹² In the “pump state” the H⁺-ATPase predominates, there is net H⁺ efflux and the hyperpolarized V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 and oscillations between these states may explain the temporal changes in H⁺ flux recently observed at the apex of growing Arabidopsis root hairs.¹⁵ Peaks of H⁺ influx may reflect a depolarized V_m that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca²⁺ influx it permits could be amplified through intracellular release.The PM H⁺-ATPase traditionally lies at the core of models of voltage and ionic flux^14,16 but in terms of [Ca²⁺]_cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.⁵ AtrbohC is implicated in the transition to polar growth at normal extracellular pH⁵ and also osmoregulation.¹⁷ NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e⁻ efflux systems,¹⁸ their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.¹⁹ Two H⁺ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e⁻ efflux by animal NADPH oxidases is fairly constant over negative V_m and decreases at very depolarized V_m.²⁰ AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H⁺ flux oscillations.¹⁵ The latter suggests that AtrbohC function does in some way affect V_m, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.²¹NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency^22,23 to signal stress conditions. Blockade of PM Ca²⁺ channels by lanthanides increases superoxide production in tobacco suspension cells.²⁴ This suggests that NADPH oxidases are involved in sensing the cell''s Ca²⁺ status and the prediction would be that extracellular Ca²⁺ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).^25,26 Lowering extracellular Ca²⁺ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca²⁺ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca²⁺]_cyt²⁷ (Fig. 1). However, reduction of Ca²⁺ influx should promote voltage hyperpolarisation (just as block of K⁺ influx causes hyperpolarisation in root hairs²⁸) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity⁵ to increase Ca²⁺ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl₂, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ to increase [Ca²⁺]_cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to V_m, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca²⁺]_cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing V_m and [Ca²⁺]_cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.²⁹ While there is still much to do on the “inventory” of PM transporters involved in Ca²⁺ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action.     

Are there multiple circadian clocks in plants?

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types.¹ Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type.² However, we found that cytosolic free Ca²⁺ ([Ca²⁺]_cyt) oscillations were undetectable in these mutants in the same light conditions.¹ Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca²⁺]_cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (∼21 h) but, surprisingly, has a wild-type period for circadian [Ca²⁺]_cyt oscillations (∼24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca²⁺]_cyt rhythms (∼21 h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations might be regulated differently.Key words: circadian rhythms, TOC1, multiple oscillators, CAB2, Ca²⁺ signalling, arabidopsis, circadian [Ca²⁺]_cyt oscillations, aequorin, luciferase, central oscillatorThe plant circadian clock controls a multitude of physiological processes such as photosynthesis, organ and stomatal movements and transition to reproductive growth. A plant clock that is correctly matched to the rhythms in the environment brings about a photosynthetic advantage that results in more chlorophyll, more carbon assimilation and faster growth.³ One of the first circadian clock mutants to be described in plants was the short period timing of cab1-1 (toc1-1), which was identified using the rhythms of luciferase under a CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter as a marker for circadian period.⁴Circadian rhythms of both CAB2 promoter activity and cytosolic-free Ca²⁺ ([Ca²⁺]_cyt) oscillations depend on the function of a TOC1, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL (TOC1/CCA1/LHY) negative feedback loop.⁵ In tobacco seedlings, CAB2:luciferase (CAB2:luc) rhythms and circadian [Ca²⁺]_cyt oscillations can be uncoupled in undifferentiated calli.⁶ In Arabidopsis, we reported that toc1-1 has different periods of rhythms of CAB2 promoter activity (∼21 h) and circadian [Ca²⁺]_cyt oscillations (∼24 h). The mutant allele toc1-1 has a base pair change that leads to a full protein that has an amino acid change from Ala to Val in the CCT domain (CONSTANS, CONSTANS-LIKE and TOC1).⁷ On the other hand, the mutant toc1-2 has short period of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations (∼21 h).^1,7 This allele has a base pair change that results in changes to preferential mRNA splicing, resulting in a truncated protein with only 59 residues.⁷ Thus, the mutated CCT domain in toc1-1 might lead to the uncoupling of rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations while the absence of TOC1 in toc1-2 causes the shortening of the period of both rhythms. Indeed, zeitlupe-1 (ztl-1) mutants, that have higher levels of TOC1, have long periods of both rhythms of CAB2 promoter activity and circadian [Ca²⁺]_cyt oscillations.¹ The biochemical function of the CCT domain is unknown but it is predicted to play an important role in protein-protein interactions⁸ and nuclear localization.⁹One model to explain the period difference of CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation is that the toc1-1 mutation has uncoupled two oscillators in the same cell. Uncoupled oscillators are a predicted outcome of certain mutations in the recently described three-loop mathematical model.^10–11 However, both rhythms of TOC1 and CCA1/LHY expression, which would be in uncoupled oscillators accordingly to the model, are described as short-period in toc1-1.⁵ Thus, we have favored the model in which CAB2:luc expression and circadian [Ca²⁺]_cyt oscillation are reporting cell-types with different oscillators that are affected differently by toc1-1.It is possible that TOC1 could interact with a family of cell-type specific proteins. The interaction of TOC1 with each member of the family could be affected differently by the mutation in the CCT domain (Fig. 1). Two-hybrid assays have shown that TOC1 interacts with PIF proteins (PHYTOCHROME INTERACTING FACTOR3 and PIF4) and related PIL proteins (PIF3-LIKE PROTEIN 1, PIL2, PIL5 and PIL6).⁸ In fact, TOC1 interaction with both PIF3 and PIL1 is stronger when the N-terminus receiver domain is taken out and the CCT domain is left intact.⁸ Thus, it is possible that TOC1 and different PIF/PIL proteins interact to regulate the central oscillator. This interaction could be impaired by the Ala to Val change in the toc1-1 mutation, leading to the period shortening. However, lines misexpressing PIF3, PIL1 and PIL6 showed no changes in their circadian rhythms.^12–16Open in a separate windowFigure 1Models of how the toc1-1 mutation might differently affect cell-type specific circadian oscillators. The single mutant toc1-1 have 21 h rhythms of CAB2 promoter activity and 24 h-rhythms of [Ca²⁺]_cyt oscillations. The toc1-1 mutation is a single amino acid change in the CCT domain. The CCT domain is involved in protein-protein interaction and/or nuclear localization. We have proposed that circadian oscillators with different periods are present in different cell-types. The luminescence generated by CAB2 promoter-drived luciferase (from the CAB2:luc) is probably originated in the epidermis and mesophyll cells. In this model, we propose that the mutation on the CCT domain impairs the mutated TOC1 interaction with the hypothetical protein Z in these cells-types. In contrast, in other cell-types, the mutated TOC1 still interacts with other hypothetical proteins (W), despite the mutation in the CCT domain. In those cell-types, the circadian oscillator could still run with a 24 h period for [Ca²⁺]_cyt rhythms (from the 35S:AEQ construct). One possible identity for Z and W are the members of the PHYTOCHROME INTERACTING FACTOR (PIF) related PIF3-LIKE (PIL) family.One possible explanation for the absence of alterations in the period of circadian rhythms in lines misexpressing PIF/PIL is that they only have roles in certain cell-types. As an example, PIL6 and PIF3 are involved with flowering time and hypocotyl growth in red light^12–15 while PIL1 and PIL2 are involved with hypocotyl elongation in shade-avoidance responses.¹⁶ Both hypocotyl growth and flowering time require cell-type specific regulation: vascular bundle cells in the case of the flowering time¹⁷ and the cells in the shoot in the case of the hypocotyl elongation.¹⁶ If TOC1 interaction with certain PIF/PIL is indeed cell-type specific, the mutated CCT domain found in the toc1-1 mutant could affect the clock in different ways, depending on the type of PIF/PIL protein expressed in each cell-type. Therefore, a question that arises is: which cell-types are sensitive to the toc1-1 mutation?There is evidence that CAB2 and CATALASE3 (CAT3) are regulated by two oscillators that respond differently to temperature signals.¹⁸ These genes might be regulated by two distinct circadian oscillators within the same tissues or a single cell.¹⁸ Interestingly, the spatial patterns of expression of CAB2 and CATALASE3 overlap in the mesophyll of the cotyledons.¹⁸ Furthermore, rhythms of CAB2 and CHALCONE SYNTHASE (CHS) promoter activity have different periods and they are equally affected by toc1-1 mutation.¹⁹ Whereas CAB2 is mainly expressed in the mesophyll cells, CHS is mainly expressed in epidermis and root cells.¹⁹ However, rhythms of AEQUORIN luminescence, which reports [Ca²⁺]_cyt oscillation, were insensitive to toc1-1 mutation and appear to come from the whole cotyledon.²⁰ One cell-type which is found in the whole cotyledon but is distinct from either mesophyll or epidermis cells is the vascular tissue and associated cells.Another approach to determine which cell-types are insensitive to toc1-1 mutation is to compare the toc1-1 and toc1-2 phenotypes. The period of circadian [Ca²⁺]_cyt oscillations is not the only phenotype that is different in toc1-1 and toc1-2 mutants. Rhythms in CAB2 promoter activity in constant red light are short period in toc1-1 but arrhythmic in toc1-2.^21,22 COLD, CIRCADIAN RHYTHM AND RNA BINDING 2/GLYCINE-RICH RNA BINDING PROTEIN 7 (CCR2/GRP7) is also arrhythmic in toc1-2 but short period in toc1-1 in constant darkness.^7,22 When the length of the hypocotyl was measured for both toc1-1 and toc1-2 plants exposed to various intensities of red light, only toc1-2 had a clear reduction in sensitivity to red light. Therefore, toc1-2 has long hypocotyl when maintained in constant red light while hypocotyl length in toc1-1 is nearly identical to that in the wild-type.²² These differences may allow us to separate which cell-types are sensitive to the toc1-1 mutation and which not.Hypocotyl growth is regulated by a large number of factors such as light, gravity, auxin, cytokinins, ethylene, gibberellins and brassinosteroids.²³ There is also a correlation between the size of the hypocotyl in red light and defects in the circadian signaling network.^24,25 The fact that toc1-1 has different hypocotyl sizes from toc1-2 suggests that circadian [Ca²⁺]_cyt oscillations could be involved in the light-dependent control of hypocotyl growth. Circadian [Ca²⁺]_cyt oscillations might encode temporal information to control cell expansion and hypocotyl growth.^26–28 toc1-1 have short-period rhythms of hypocotyl elongation, which indicates that the cells in the hypocotyl have a 21 h oscillator.²⁹ However, toc1-1 might also have a wild-type hypocotyl length in continuous red light because cells which generate the signal to regulate hypocotyl growth might have 24 h oscillators.The toc1-1 mutation was the first to be directly associated with the plant circadian clock, revitalizing the field of study.⁴ Now, by either uncoupling two feedback loops or by distinct TOC1 protein-protein interaction in different cell-types, toc1-1 has shown new properties of the circadian clock that may deepen our understanding of this system.     

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

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Extracellular Nucleotides Elicit Cytosolic Free Calcium Oscillations in Arabidopsis

Extracellular ATP induces a rise in the level of cytosolic free calcium ([Ca²⁺]_cyt) in plant cells. To expand our knowledge about the function of extracellular nucleotides in plants, the effects of several nucleotide analogs and pharmacological agents on [Ca²⁺]_cyt changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca²⁺ sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca²⁺]_cyt that displayed distinct time- and dose-dependent kinetics compared with the purine nucleotides ATP and GTP. The inhibitory effects of antagonists of mammalian P2 receptors and calcium influx inhibitors on nucleotide-induced [Ca²⁺]_cyt elevations were distinct between CTP and purine nucleotides. These results suggest that distinct recognition systems may exist for the respective types of nucleotides. Interestingly, a mutant lacking the heterotrimeric G protein Gβ-subunit exhibited a remarkably higher [Ca²⁺]_cyt elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gβ in negatively regulating extracellular nucleotide signaling and point to an important role for heterotrimeric G proteins in modulating the cellular effects of extracellular nucleotides. The addition of extracellular nucleotides induced multiple temporal [Ca²⁺]_cyt oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicle-trafficking inhibitor, indicating that the oscillations likely require ATP release via exocytotic secretion. The results reveal new molecular details concerning extracellular nucleotide signaling in plants and the importance of fine control of extracellular nucleotide levels to mediate specific plant cell responses.The calcium ion, Ca²⁺, is a ubiquitous second messenger that is used to regulate a wide range of cellular processes (Clapham, 2007). A number of plant environmental and developmental responses are encoded to distinct Ca²⁺ signal patterns with specific frequencies and amplitudes of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). These signal patterns can take the form of pulsating [Ca²⁺]_cyt spiking/oscillations (Berridge et al., 2003). In plants, such [Ca²⁺]_cyt oscillations occur in various cell types (e.g. stomatal guard cells, pollen tubes, and legume root hairs) and play a critical role in responding to environmental signals (Evans et al., 2001; Oldroyd and Downie, 2008; McAinsh and Pittman, 2009).ATP is a ubiquitous compound in all living cells; it not only provides the energy to drive many biochemical reactions but also functions in signal transduction as a substrate for kinases, adenylate cyclases, etc. However, ATP was also shown to be an essential signaling agent outside of cells in animals, where it is referred to as extracellular ATP. Extracellular ATP is involved in numerous cellular processes, including neurotransmission, immune responses, cell growth, and cell death (Khakh and Burnstock, 2009). In mammalian cells, plasma membrane-localized receptors, purinoceptors of the P2X and P2Y classes, bind ATP as well as other nucleotides at the cell surface to activate intracellular signaling cascades via second messengers. Binding of extracellular ATP to P2X receptors gates calcium influx, whereas activation of P2Y receptors stimulates the recruitment of heterotrimeric G proteins to trigger cytoplasmic signaling and gene expression. As a common phenomenon, the activated receptors induce the elevation of [Ca²⁺]_cyt, which in turn activates the production of downstream messengers such as nitric oxide and reactive oxygen species (ROS; Shen et al., 2005; Fields and Burnstock, 2006).A possible physiological role for extracellular ATP in plants was first reported in studies in which exogenously applied ATP was found to stimulate closure of the Venus flytrap (Dionaea muscipula; Jaffe, 1973), to induce the formation of nucleases in excised Avena leaves (Udvardy and Farkas, 1973), and to induce potassium ion uptake into cells of maize (Zea mays) leaf slices (Lüttge et al., 1974). Over the past several years, extracellular ATP was found to be an important signaling compound in plants that induces various plant responses, including root-hair growth (Lew and Dearnaley, 2000; Kim et al., 2006), stress responses (Thomas et al., 2000; Jeter et al., 2004; Song et al., 2006), gravitropism (Tang et al., 2003), cell viability (Chivasa et al., 2005), pathogen responses (Chivasa et al., 2009), and thigmotropism (Weerasinghe et al., 2009). The release of extracellular ATP from root cells was directly imaged by Kim et al. (2006) using a luciferase construct engineered to bind to plant cell wall cellulose. Recently, using this reporter, Weerasinghe et al. (2009) measured the release of ATP from root cells in response to touch. This documentation of the presence of extracellular ATP in plants at levels sufficient to induce cellular responses suggests that extracellular ATP likely plays an important role throughout plant growth and development. However, no P2 receptor homologs have been identified in plants, despite the fact that plants share a number of cellular responses to ATP with animal cells. For example, the addition of exogenous ATP or ADP triggers an increase in [Ca²⁺]_cyt levels in whole seedlings, dissected root tissues, and root epidermal protoplasts of Arabidopsis (Arabidopsis thaliana; Demidchik et al., 2003, 2009; Jeter et al., 2004). The production of ROS in response to ATP addition was detected in various plant tissues (Kim et al., 2006; Song et al., 2006; Wu et al., 2008; Demidchik et al., 2009). More recently, the plasma membrane NADPH oxidase RBOHC (for respiratory burst oxidase homolog C) was shown to be required for extracellular ATP-induced ROS production in Arabidopsis primary roots (Demidchik et al., 2009). Extracellular ATP also stimulates the production of nitric oxide in tomato (Solanum lycopersicum) culture cells and in Salvia miltiorrhiza hairy roots (Foresi et al., 2007; Wu and Wu, 2008). These reports suggest that extracellular ATP signals across the plasma membrane by triggering elevation in [Ca²⁺]_cyt, which activates the production of downstream messengers. Ultimately, these cell responses induce the expression of various genes, such as MAPKs, LOX, and ACS6 (Jeter et al., 2004; Song et al., 2006), and cause physiological responses, as described above.In animal cells, extracellular ATP-evoked elevations in [Ca²⁺]_cyt are often observed in the form of oscillations that result from the transient opening of Ca²⁺ channels located either in the plasma membrane or in cytosolic Ca²⁺ stores. Intracellular calcium release is often mediated through phospholipase C (PLC)-mediated signaling coupled to heterotrimeric G proteins (Mahoney et al., 1992; Visegrady et al., 2000; Hanley et al., 2004). In plants, plasma membrane Ca²⁺-permeable channels are known to contribute to extracellular ATP-induced [Ca²⁺]_cyt elevation (Demidchik et al., 2009). However, neither the mechanisms underlying extracellular ATP-evoked Ca²⁺ signaling nor the possible involvement of heterotrimeric G proteins has been characterized in plants.In order to explore their roles as possible ligands of putative nucleotide receptors, the plant [Ca²⁺]_cyt response to six different nucleotides (Fig. 1A) was measured using Arabidopsis seedlings expressing one of two [Ca²⁺]_cyt sensors, either aequorin or the fluorescence resonance energy transfer (FRET)-based Ca²⁺ sensor Yellow Cameleon 3.6 (YC3.6). The pyrimidine nucleotide CTP as well as the purine nucleotides ATP and GTP induced a strong elevation of [Ca²⁺]_cyt in seedlings. Interestingly, the effects of all the nucleotides on Ca²⁺ signaling were negatively regulated by a heterotrimeric G protein β-subunit, AGB1. The addition of ATP to aequorin-expressing seedlings induced distinct [Ca²⁺]_cyt oscillations in the presence of the apyrase inhibitor NGXT191. However, in the absence of this inhibitor, such [Ca²⁺]_cyt oscillations could be localized to specific root cell layers using YC3.6 fluorescence. Given the importance of [Ca²⁺]_cyt oscillations in intracellular signaling, the data suggest an important, unexplored role of extracellular ATP in the plant signaling pathways.Open in a separate windowFigure 1.NTPs increase bioluminescence in aequorin-expressing transgenic Arabidopsis seedlings. A, Chemical structures of purine and pyrimidine derivatives. B, Individual 5-d-old aequorin seedlings were transferred to individual wells of a 96-well microplate and incubated overnight in reconstitution buffer containing coelenterazine. Each NTP was then applied at a final concentration of 100 μm. The line graph shows time-dependent changes in photon counts from representative wells of each treatment (bin size = 50 frames, 1 s, 20 bin smoothing). The inset shows a pseudocolored photon-counting image integrated over 400 s after nucleotide treatment calibrated to the inset scale.     

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

Depolarization-evoked synaptic transmission relies on the Ca²⁺-regulated release of quantal packets of neurotransmitters following the fusion of synaptic vesicles with the presynaptic plasma membrane. It is well known that neuronal voltage-gated Ca²⁺ channels (VGCC), mainly of the Ca_V2.1 and Ca_V2.2 subtypes, play a key role in the first steps of this process, by controlling extracellular Ca²⁺ influx into active zones of the synapse. These channels are in close association with the vesicle machinery and interact with several members of SNARE proteins (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) including syntaxin 1A/1B and SNA P-25 (Q-SNARE s), and synaptotagmin 1 and synaptobrevin 2 (R-SNARE s) (reviewed in ref. ¹). All bind to the synprint (synaptic protein interaction) motif within the intracellular II -III linker of Ca_V2.1 and Ca_V2.2 channels and are responsible for a bidirectional coupling (i) linking the Ca²⁺ influx with the synaptic vesicle release machinery, which is essential for efficient, fast and spatially delimited neurotransmitter release² and (ii) providing regulation of Ca²⁺ channel activity and thus of Ca²⁺ influx.³Key words: calcium channel, Ca_V2.1 channel, P/Q channel, syntaxin, synaptotagmin, SNAP25, exocytosis, synaptic transmissionSeveral studies have proposed that synaptotagmin 1 is the Ca²⁺ sensor for release, linking Ca²⁺ influx to vesicle fusion (reviewed in ref. ⁴). Synaptotagmin 1 has two repeating domains that are rich in negative charges (C2A and C2B), each capable of binding Ca²⁺ ions. It is commonly thought that following Ca²⁺ entry through VGCCs, Ca²⁺ ions bind to C2A and C2B domains, allowing insertion of the Ca²⁺ binding loops of C2A domain in the target bilayer. This then pins the vesicle to the plasma membrane to trigger exocytotic fusion. This view was supported by a point mutation in the C2A domain of synaptotagmin 1 that caused a decrease in Ca²⁺ affinity with a concomitant decrease of neurotransmitter release.⁵ However, despite the fact that synaptotagmin 1 represents the most popular candidate for Ca²⁺ sensor, the initial Ca²⁺ binding event, which occurs during the dynamic process of release is at the EEEE locus within the Ca²⁺ channel itself. This makes the Ca²⁺ channel an excellent candidate for serving as a Ca²⁺ sensor of secretion.⁶Over the past few years, the group of Daphne Atlas has performed extensive studies to differentiate the role of Ca²⁺ binding at the pore of the channel from Ca²⁺ binding to intracellular proteins during evoked-neurotransmitter release. Substituting extracellular Ca²⁺ by lanthanum (La³⁺), a trivalent cation that effectively binds to the EEEE locus of VGCCs but is unable to permeate through the channel, is sufficient to support depolarization-evoked release of catecholamine in PC12 and primary chromaffin cells, as well as insulin release in pancreatic and insulinoma cells. These results led to the suggestion that evoked release may be dependent on ion channel pore occupancy as opposed to cation influx and elevation of intracellular Ca²⁺ concentration.^7–9 This model was further supported by experiments in which depolarization-evoked secretion of catecholamine in chromaffin cells was supported by Ca²⁺ bound at the selectivity filter of a non-conducting Ca_V1.2 channel.¹⁰ These studies are consistent with the proposal that conformational changes subsequent to Ca²⁺ binding at the selectivity filter of the channel are the primary trigger of secretion, whereas synaptotagmin 1 is associated with the channel and acts as a vesicle docking protein (reviewed in ref. ¹¹).In a recent issue of Channels, Cohen-Kutner et al. extended this concept to the neuronal Ca_V2.1 channel.¹² Using the two-electrode voltage-clamp technique on BAPTA-injected Xenopus oocyte expressing the human Ca_V2.1 channel (in combination with β₃ and α₂δ auxiliary subunits), the authors show that overexpression of syntaxin 1A (Stx1A) depresses whole-cell inward barium (Ba²⁺) current in a dose-dependent manner (Fig. 1, reviewed in ref. ¹²). As previously reported by Bezprozvanny et al.³ this effect is mainly due to a hyperpolarized shift of the steady-state inactivation curve, which decreases the number of available channels at typical resting membrane potentials. A recovery of channel activity is observed following co-expression of botulinium neurotoxin C1 (BoNT/C1) (Fig. 3, reviewed in ref. ¹²). In contrast, expression of the other Q-SNARE protein SNAP-25 drastically increases inward Ba²⁺ current (Fig. 2, reviewed in ref. ¹²). However, when both Q-SNARE proteins are co-expressed, Ca_V2.1 channel recovers wild-type P/Q kinetics and current amplitude (Fig. 2, reviewed in ref. ¹²). Similarly, increases in P/Q currents by expressing the R-SNARE synaptobrevin (VAMP-2) are reversed by the Q-SNARE proteins (Fig. 4, reviewed in ref. ¹²). Taken together these results suggest that: (i) when expressed in BAPTA injected Xenopus oocyte, each of the SNARE proteins is able to modulate the kinetic properties of Ca_V2.1 channel and (ii) when co-expressed, SNARE proteins no longer affect channel activity but rather form a Ca²⁺-independent excitosome complex with a fully functional channel. These data fit nicely with previous work from the Catterall laboratory on P/Q-type channels,¹³ and with previous work on N-type channels.¹⁴To investigate the relevance of Ca_V2.1 channel interaction with SNARE proteins for depolarization-evoked secretion, membrane capacitance changes induced in Xenopus oocytes were monitored in the presence of extracellular Ca²⁺, as previously shown for Ca_V1.2 and Ca_V2.2.¹⁵ While expression of Ca_V2.1 alone in this reconstituted release assay produced only a small change in capacitance, coexpression with the SNARE proteins efficiently induced a BoNT/C- and BoNT/A-sensitive membrane fusion, particularly when all SNARE proteins were co-expressed, i.e., when all members of the excitosome complex are present (Fig. 5, reviewed in ref. ¹²). Hence, increasing the amount of excitosome promotes the capability of Ca_V2.1 channels to produce evoked-secretion, probably by increasing the number of functional excitosome complexes (Fig. 6, reviewed in ref. ¹²).In summary, Cohen-Kutner et al. provide evidence that when expressed in Xenopus oocyte (and possibly in other cellular systems), Ca_V2.1 channels could associate with SNARE proteins at resting intracellular Ca²⁺ concentrations, resulting in tethering the vesicle to the channel and thereby generating docked but non-releasable vesicles. Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Syt1 C2 domains. The fusion of releasable vesicles requires a conformational change of the complex that occurs within the channel itself, during an incoming action potential (Fig. 1).Open in a separate windowFigure 1A putative model of functional coupling between Ca_V2.1 channel and vesicle release machinery. At resting membrane potential, Ca_V2.1 channel associates with SNARE proteins to form an excitosome complex, in turn generating docked but non-releasable vesicle (A). Calcium entry following membrane depolarization would switch the vesicle from the non-releasable to a releasable state by Ca²⁺-binding to Synaptotagmin 1 C2 domains (B). The fusion of the releasable vesicle requires a conformational change of the excitosome complex that occurs within the channel itself, during an incoming action potential (C).The concept that Ca_V2.1 channels, besides sustaining Ca²⁺ influx, could also work as a molecular on/off-switch of secretion by controlling the ultimate stage of the process (i.e., the conformational change of the releasing complex) is intriguing and is worthy of further investigation. To better dissociate secretion events linked to Ca²⁺ entry through Ca_V2.1 channel from those induced by conformational changes of the channel, it would be necessary to measure secretion in the presence of a non-permeant cation such as La³⁺. Furthermore, one would also need to evaluate mediation of secretion by a non-conducting Ca_V2.1 channel, as already done for L-type channels (Ca_V1.2).^7,9,10 Moreover, the possibility that Ca_V2.1 channels could control secretion via a conformational change of the releasing complex raises questions concerning the preferential channel-gating mode controlling this process. It was recently shown that application of the gating modifier BayK 8644 to non-conducting Ca_V1.2 channels modifies secretion kinetics of catecholamine in chromaffin cells.¹⁶ It is also well known that the auxiliary β-subunit of VGCCs modulates Ca_V2.1 gating modes.¹⁷ Therefore, comparing secretion mediated by a non-conducting Ca_V2.1 channel in the presence of different types of β-subunits would provide important information on the molecular mechanisms through which Ca_V2.1 channels control evoked-secretion, both at the fundamental and physiopathological levels.In conclusion, since the pioneering work by Katz and Miledi in 1967 on the importance of the extracellular Ca²⁺ in the “electro-secretory” process,¹⁸ the identification of the calcium channel as the Ca²⁺ sensor of secretion is one of the most recent and exciting steps that have been made in the understanding of the molecular aspects of the mechanisms involved in the control of depolarization-evoked neurotransmitter release.     

Step by Step: Deciphering Ion Transport in the Root Xylem Parenchyma

Proton pumps produce electrical potential differences and differences in pH across the plasma membrane of cells which drive secondary ion transport through sym- and antiporters. We used the patch-clamp technique to characterize an H⁺-pump in the xylem parenchyma of barley roots. This cell type is of special interest with respect to xylem loading. Since it has been an ongoing debate whether xylem loading is a passive or an active process, the functional characterization of the H⁺-pump is of major interest in the context of previous work on ion channels through which passive salt efflux into the xylem vessels could occur. Cell-type specific features like its Ca²⁺ dependence were determined, that are important to interpret its physiological role and eventually to model xylem loading. We conclude that the electrogenic pump in the xylem parenchyma does not participate directly in the transfer of KCl and KNO₃ to the xylem but, in combination with short-circuiting conductances, plays a crucial role in controlling xylem unloading and loading through modulation of the voltage difference across the plasma membrane. Here, our recent results on the H⁺ pump are put in a larger context and open questions are highlighted.Key Words: plant nutrition, H⁺-ATPase, anion conductance, K⁺ channel, electrophysiology, signaling networkThe root xylem parenchyma is of major interest with respect to nutrient (and signal) traffic between root and shoot. One of its main functions appears to be xylem loading. However, the cell walls of the vascular tissue provide apoplastic paths between xylem and phloem that represent the upward and downward traffic lanes, allowing nutrient circulation¹ (Fig. 1). Therefore mechanisms for ion uptake and for ion release must exist side by side. In the last 15 years major progress has been made in the investigation of transport properties of xylem-parenchyma cells, and both uptake and release channels and transporters were identified. Today, we have good knowledge on the role of K⁺ and anion conductances in xylem loading with salts.² Note, that from the functionally well characterized conductances only the molecular structure of K⁺ channels is known. In contrast, many transporters are identified on the molecular level, but functional data are scarce.Open in a separate windowFigure 1Distribution of tissues in the periphery of the stele. The stippled area marks the region from which early metaxylem protoplasts originated. E, Endodermis with Casparian strip; eMX, ‘early’ metaxylem vessel; IMX, ‘late’ metaxylem vessel; Mph, metaphloem (sieve tube); Pph, protophloem (sieve tube); P, pericycle; Cx, cortex. Symplasmic and apoplasmic transport routes are indicated in red and black, respectively. The Casparian strip prevents apoplastic transport into the stele. Plasmodesmata are shown exemplarily for the indicated symplastic pathway. All cells of the symplast are connected via plasmodesmata. Sites of active uptake into the root symplast and of release into the stelar apoplast are indicated by a black and an orange arrow. Modified from Wegner and Raschke, 1994.³A challenging question to deal with was the dispute about xylem loading with ions being a passive or active process. While it is clear that energy through electrogenic H⁺ efflux is needed to take up nutrient ions from the soil against their electrochemical gradient into the cortical symplast, it has been a matter of debate if ion release into xylem vessels also is energy-linked or if the electrochemical potentials of ions are raised high enough to allow a thermodynamically passive flux.^2,3 The Casparian strip prohibits apoplastic transport of nutrients into the stele and electrically insulates the stelar from the cortical apoplast. Therefore the electrical potential difference of the cells in the xylem parenchyma could be independent from the cortical potential difference but be subject to control, for instance, from the shoot.⁴ Indeed, evidence points to xylem loading as a second control point in nutrient transfer to the shoot.^5,6 The identification and characterization of K⁺ and anion conductances clearly showed that release of KCl and KNO₃ into the xylem can be passive through voltage-dependent ion channels.^2,3,7–9 No need appeared for a pump energizing the transfer of salts to the xylem.However, H⁺ pumps are ubiquitous. H⁺-ATPases are encoded by a multigene family and heterologous expression in yeast showed that isoforms have distinct enzymatic properties.^10,11 As the example of the amino acid transporter AAP6 from the xylem parenchyma shows, a cell-type specific functional characterization of transporters is essential to draw conclusions on their physiological role. AAP6 is the only member of a multigene family with an affinity for aspartate in the physiologically relevant range. The actual apoplastic concentration of amino acids and the pH will determine what is transported in vivo.^12,13 Xylem-parenchyma cells of barley roots were strongly labelled by antibodies against the plasma membrane H⁺-ATPase.¹⁴ In a recent publication in Physiologia Plantarum we report the functional analysis of the electrogenic pump from the plasma membrane of xylem parenchyma from barley roots that was done with the patch-clamp technique after specific isolation of protoplasts from this cell type. It displayed characteristics of an H⁺-ATPase: current-voltage relationships were characteristic for a ‘rheogenic’ pump¹⁵ and currents were stimulated by fusicoccin or by an enlarged transmembrane pH gradient and inhibited by dicyclohexylcarbodiimide (DCCD). Importantly, it also showed distinct characteristics. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Noteworthy, K⁺ and anion conductances from the same cell type are controlled by intracellular [Ca²⁺]^7,9 (Fig. 2). It was proposed that the effect of abscisic acid (ABA) on anion conductances is mediated via an increase in the cytosolic Ca²⁺ concentration.¹⁶ Very likely stelar H⁺ pumps are stimulated by ABA.¹⁷ Thus, a Ca²⁺ independent control has to be hypothesized in this case.Open in a separate windowFigure 2Control of ion conductances in the plasma membrane of xylem-parenchyma cells. Arrowheads indicate stimulation and bars indicate inhibition by an increase in cytosolic [Ca²⁺],^7,9,16 by ABA,^16,17,21 by cytosolic and apoplastic acidification,^4,22 by G-proteins²³ and by an increase in apoplastic [K⁺]⁷ and [NO₃⁻].²⁴ Apoplastic [K⁺] and [NO₃⁻] modify the voltage dependence exerting negative feedback on K⁺ efflux and a positive feedback on NO₃⁻ efflux. Abscisic acid has an immediate effect on ion channel activity, most likely via [Ca²⁺], and causes a change in gene expression as indicated by circles (up) and bars (down). ABA perception is not clear. A Ca²⁺ influx could occur through a hyperpolarization activated cation conductance (HACC).^16,25 Cation transporters are NORC, nonselective cation conductance, KORC, K⁺-selective outwardly rectifying conductance (=SKOR⁸), and KIRC, K⁺-selective inwardly rectifying conductance, and anion conductances with different voltage-dependencies and gating characteristics are X-QUAC, quickly activating anion conductance, X-SLAC, slowly activating anion conductance, and X-IRAC, inwardly rectifying anion channel.^2,3,9,16,26 Transported ions and direction of flux are plotted.To date, we know that besides Ca²⁺ and abscisic acid also the pH, nonhydrolyzable GTP analogs and extracellular NO₃⁻ and K⁺ affect membrane transport capacities of root xylem-parenchyma cells (Fig. 2). Other control mechanisms by metabolites, the redox potential and phytohormones have to be included, especially if they represent signals in xylem loading or root-shoot communication. The composition of the xylem sap changes during the course of a day, depending on nutrient supply and various stresses, and the apoplastic ion concentration is considered to be an important factor in ion circulation.^6,18,19 ABA is such a signal. It is known to increase solute accumulation within the root by inhibiting release of ions into the xylem.¹⁷ Any change in transport activity has an impact on the membrane potential. This again determines whether salt release or uptake takes place. Passive salt release is restricted to a limited range of membrane potentials in which conductances for anions and cations are active simultaneously, that is with depolarization. Negative membrane voltages will be required for reabsorption of NO₃⁻ by a putative NO₃⁻/H⁺-symporter and for the uptake of K⁺ and amino acids.^3,13 As shown in our recent paper, the balance between the activities of the H⁺-pump and the anion conductances could affect the position between a depolarized and a hyperpolarized state of the parenchymal membrane. Thus, H⁺ pump activity is crucial in membrane voltage control. Furthermore, the simultaneous activities of H⁺ pumps and anion conductances make the generation of a high pH gradient possible, whilst maintaining electroneutrality. The proton gradient could be used for ion transport through cotransporters and antiporters as suggested for the loading of borate into the xylem through the boron transporter BOR1.²⁰ So we are on the way to decipher xylem loading in roots and this exciting field will also provide information about small-scale nutrient cycling and root-shoot communication. To determine how the activities of pumps, channels and transporters are adjusted among each other is the next challenge. Further insight has to be obtained by experimentation as well as by biophysical modeling.     

Lily Cdc42/Rac-interactive binding motif-containing protein,a Rop target,involves calcium influx and phosphoproteins during pollen germination and tube growth

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca²⁺ influx blocking agents LaCl3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl₃ and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca²⁺ influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl₃ and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca²⁺]_cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.Key words: calcium influx, phosphoprotein, pollen tube growth, RIC protein, rop GTPaseRop (Rho GTPase of plants) was newly reported as a master regulator for plant signaling.^1,2 It participates in concerted actions of many signaling pathways that influence growth and development, and the adaptation of plants to various environmental situations.^3–5 In contrast to be negative regulators in ABA signaling,⁶ Rops might work as positive regulators in auxin signaling pathways.^7,8 Recently, we have reported unique desiccation-associated ABA signaling in which the LLP-Rop1 gene is not only negatively regulated by desiccation but also positively regulated by developmental cues independent of ABA during pollen maturation.⁹ Although LLP-Rop1 and its target, LLP12-2, accumulate in abundance in the matured and dried pollen upon dehydration, the activity of LLP-Rop1 and LLP12-2 is likely restricted at the stage of pollen maturation.⁹ As pollen germinates, ABA content decreases its level in the growing tube and thus, the activity of Rop is less restricted than that in the dried pollen and subsequently Rops become powerful regulators playing crucial roles during pollen tube growth.Pollen germination and tube growth are a continuous and highly polarized process characteristic of tip growth. As soon as pollen hydrates and germinates, a tip-focused cytoplasmic Ca²⁺ gradient is established and sustained while a pollen tube grows forward.^10,11 The Ca²⁺-permeable channels that modulate [Ca²⁺]_cyt influx in germinating pollen grains have been identified in Arabidopsis,^12,13 lily¹⁴ and pear.¹⁵ When a pollen tube grows, Rop-interactive Cdc42/Rac-interactive binding (CRIB) motif-containing proteins (RICs) play an important role as Rop GTPase targets and control a variety of Rop-dependent signaling pathways.¹⁶ RICs contain a CRIB motif required for their specific interaction with GTP-bound Rop. They are grouped into five classes that share little sequence similarity outside of the Rop-interactive domain.¹⁶ Different RICs expressed in various reproductive and vegetative parts of the Arabidopsis plant may act as Rop targets to control different Rop-dependent pathways in pollen tubes and in other organ development. For instances, RIC4 has been demonstrated to promote F-actin assembly, whereas RIC3 activates Ca²⁺ signaling by affecting [Ca²⁺]_cyt influx that subsequently results in F-actin disassembly in pollen tube growth.¹⁷ The two RICs, both activated by AtRop1, counteract each other to control the actin dynamics and polar pollen tube growth.¹⁷We have demonstrated that LLP12-2, a RIC protein, interacts with active LLP-Rop1 in vivo.⁹ To examine the function of LLP12-2 in the growing tubes, the purified LLP12-2 PCR product digested with XbaI and SacI was cloned into the corresponding sites of Zm13::GFP construct to generate the Zm13::GFP-LLP12-2 construct. The transient expression of GFP-LLP12-2 in pollen using particle bombardment was investigated. Pollen germination and tube length were measured after particle bombardment and subsequent in vitro germination. With the treatment of Ca²⁺ influx blocking agents LaCl₃ and EGTA, pollen expressing GFP alone significantly decreased germination and tube elongation, suggesting that a decrease in [Ca²⁺]_cyt influx may cause the inhibition of pollen germination and tube growth (Fig. 1 and 17Open in a separate windowFigure 1LLP12-2 inhibits pollen germination by regulating the calcium influx channels. Germination percentages were determined 9 h after particle bombardment and subsequent in vitro germination. Equal amounts of GFP and LLP12-2 DNA (7.5 µg) were transiently expressed in lily pollen after which pollen was treated without (control) or with either LaCl₃ (1 µM), EGTA (0.5 mM), LatB (0.05 nM), okadaic acid (5 nM) or staurosporine (1 µM) during germination.

Table 1

Effects of LaCl₃, EGTA, LatB, okadaic acid or staurosporine on the length of pollen tube expressing LLP12-2

Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca2+ exchangers

Tonoplast-localised proton-coupled Ca²⁺ transporters encoded by cation/H⁺ exchanger (CAX) genes play a critical role in sequestering Ca²⁺ into the vacuole. These transporters may function in coordination with Ca²⁺ release channels, to shape stimulus-induced cytosolic Ca²⁺ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant''s stress response. A common feature of these mutants was the perturbation of H⁺-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca²⁺/H⁺ exchangers and H⁺ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca²⁺ flux. These results suggest cautious interpretation of mutant Ca²⁺/H⁺ exchanger phenotypes that may be due to either perturbed Ca²⁺ or H⁺ transport.Key words: abiotic stress, Ca²⁺ transport, Ca²⁺/H⁺ exchanger, H⁺-ATPase, Na⁺ transport, pH, salt stress, vacuoleCa²⁺ plays a fundamental role in the plant cell, functioning as a highly versatile second messenger controlling a multitude of cellular reactions and adaptive responses.^1,2 Ca²⁺ dynamics are maintained by precise interplay among transporters involved in its release from or uptake into Ca²⁺ stores. The vacuole, as the largest internal Ca²⁺ pool, is assumed to play a major role in Ca²⁺ signalling, and has been shown to be the source of Ca²⁺ release following various abiotic stresses such as cold and osmotic stress.^3,4 Rapid, stimulus-induced release of Ca²⁺ from the vacuole is attributable to selectively permeable Ca²⁺ channels, however, the identity of candidate genes encoding this mechanism remains contested.^5,6 Better understood, are the two major vacuolar uptake mechanisms; P-type Ca²⁺ pumps, including ACA4 and ACA11, which mediate high-affinity Ca²⁺ uptake, and a family of cation/H⁺ exchangers (CAX), responsible for lower-affinity but high-capacity Ca²⁺ uptake.^7,8 While Ca²⁺ pumps rely directly on the hydrolysis of ATP to drive Ca²⁺ uptake, Ca²⁺/H⁺ exchangers are energized indirectly by the pH gradient generated by electrogenic H⁺ pumps located on the tonoplast, including the vacuolar-type H⁺-ATPase (V-ATPase).⁹With the aim of further understanding the role of specific CAX isoforms in Arabidopsis, we and others have recently characterized CAX mutants and overexpression lines and observed a variety of phenotypes, including altered response to abiotic stresses.^10–14 While some phenotypes are identical among different CAX mutants, others are specific to individual lines.¹⁴ Moreover, these analyses have highlighted the interplay of these transporters with H⁺ pumps at both the tonoplast and the plasma membrane. Overexpression of CAX1 in Arabidopsis results in increased activity of the V-ATPase, whereas mutations in CAX1 cause a concomitant decrease in measured V-ATPase activity (Fig. 1).¹¹ Similar reductions in V-ATPase activity are also observed in cax2 and cax3 mutant plants but to a lesser extent,^12,13 and a significant reduction is observed in a cax1 cax3 double knockout line.¹³ At the plasma membrane, P-type H⁺-ATPase (P-ATPase) activity is increased in cax1 but decreased in cax3 (Fig. 1).¹⁴ Indeed cax3 lines appeared more sensitive to changes in the pH of the growth media.¹⁴ This implies that unlike cax1, cax3 is less efficient at cytoplasmic pH adjustment. Another intriguing observation is that activity of the H⁺-pyrophosphatase (H⁺-PPase) at the tonoplast is largely unaltered following CAX gene deletion. While overexpression of the Arabidopsis H⁺-PPase AVP1 leads to increased Ca²⁺/H⁺ exchange activity,^15,16 there is little alteration in H⁺-PPase activity following perturbed expression of CAX1 or CAX2.^11,12 Thus, this feedback interplay appears to exist only between exchangers and H⁺-ATPases.Open in a separate windowFigure 1Tonoplast H⁺-ATPase (V-ATPase) activity and plasma membrane H⁺-ATPase (P-ATPase) activity in wild type Arabidopsis (ecotype Col-0) and Arabidopsis lines with manipulated tonoplast Ca²⁺/H⁺ exchange activity. 35S::CAX1 and 35S::CAX2 denote lines that overexpress a constitutively active N-terminally truncated CAX1 or CAX2 construct driven by the CaMV 35S promoter in the cax1-1 or cax2-1 mutant background, respectively. V-ATPase H⁺-transport activity was measured by the ATP-dependent quenching of quinacrine fluorescence, and rates of bafilomycin-sensitive, vanadate-resistant hydrolytic activity of the V-ATPase were determined in isolated tonoplast membranes, as described in refs. ¹¹ and ¹³. Rates of vanadate-sensitive, bafilomycin- and azide-resistant hydrolytic activity of the P-ATPase were determined in isolated plasma membranes, as described in ref. ¹⁴. Results are shown as % of wild type (Col-0) ATPase activity and are means ± SE of 3–4 independent experiments. Data taken and modified from refs. ^11–14.The V-ATPase is important not only for maintenance of a pH gradient across the tonoplast, but also in maintenance of Golgi structure, endocytosis and secretory trafficking.^17,18 The V-ATPase is localised at the Golgi, endoplasmic reticulum and endosomes, in addition to the tonoplast.⁹ The det3 mutant, with a mutation in subunit C (VHA-C), has a 40–60% reduction in V-ATPase activity, but numerous severe developmental phenotypes.¹⁹ In contrast, the cax1 and cax1 cax3 mutants have a reduction in V-ATPase activity equivalent to det3 (Fig. 1), but the morphological phenotypes are not as pronounced.¹³ It is therefore likely that reduction of tonoplast Ca²⁺/H⁺ exchange primarily affects tonoplast V-ATPase activity, while V-ATPase activity in the secretory pathway is unperturbed. The V-ATPase is a multi-subunit protein and some of these subunit gene products appear to be either tonoplast-specific or tonoplast-enriched. Mutations in tonoplast subunits may cause defective V-ATPase activity only at the tonoplast.⁹ It will be of interest to see whether such tonoplast-specific V-ATPase mutants phenocopy the cax mutants, and possess perturbed Ca²⁺/H⁺ exchange activity and altered abiotic stress responses.CAX-mediated transport may alter both cytoplasmic and lumenal pH, as well as intracellular Ca²⁺ gradients. In the case of the V-ATPase, evidence is emerging for a role not only in the generation of a pH gradient across membranes, but also in the direct sensing of pH within the compartment,^20,21 creating a feedback mechanism which regulates pump activity. Thus, in cax1 lines, abnormal acidification of the lumen is detected by the V-ATPase resulting in a dampening of its activity. This would conserve ATP, which we postulate could be utilized to drive the tonoplast Ca²⁺ pump which itself is upregulated in cax1 as a compensatory mechanism to correct perturbations in the Ca²⁺ gradient.¹¹ In the case of cax1, this in turn may signal the P-ATPase to remove surplus H⁺ from the cytoplasm, triggering its upregulation (Fig. 1). However, not all CAX mutants show this complex H⁺ feedback mechanism.Co-ordinate downregulation of the V-ATPase in the cax1 mutant lines may also be a result of activity of the SOS2 kinase. This Ser/Thr kinase, which specifically interacts with the N-terminus of CAX1 resulting in Ca²⁺/H⁺ exchange activation,²² upregulates V-ATPase activity through interactions with the VHA-B regulatory subunit.²³ Loss of CAX1 may be signalling to the V-ATPase through changes in SOS2 activity resulting in a compensatory downregulation of the pump. It is tempting to speculate that SOS2 may signal the alteration in P-ATPase activity, as it is known to regulate other plasma membrane proteins, notably the Na⁺/H⁺ exchanger SOS1.²⁴ It will be interesting to determine if SOS2 and the P-ATPase interact directly. It is notable, however, that SOS2 does not appear to interact with CAX3,²² while P-ATPase activity is reduced in cax3 plants.¹⁴Our recent results indicate there are at least two modes by which Ca²⁺/H⁺ exchangers can mediate adaptive responses to stress: direct manipulation of cytosolic Ca²⁺ and indirect feedback of H⁺ flux (Fig. 2). For example, salt stress responses are likely controlled via the generation of a specific cytosolic Ca²⁺ signature, which mediates a downstream signalling pathway. CAX3 appears to be the principle isoform providing tonoplast Ca²⁺/H⁺ exchange in response to salt stress.¹⁴ Disruption of CAX3-mediated tonoplast Ca²⁺ transport and the alteration of cytosolic Ca²⁺ dynamics may therefore alter the plant''s normal response to salt stress (Fig. 2). Maintenance of H⁺ gradients at both the vacuole and plasma membrane are also critical for salt tolerance, such that salt treatment upregulates V-ATPase and P-ATPase activity.²⁵ This energizes Na⁺ efflux from the cytosol mediated by Na⁺/H⁺ exchangers at the plasma membrane and the tonoplast.^24,26 Therefore downregulation of H⁺ pumps at both membranes in the cax3 mutant is likely to perturb the ability of the cell to remove Na⁺ (Fig. 2). Further analysis of cax mutants, P-ATPase mutants, and tonoplast-specific V-ATPase mutants will be required to determine whether many of the phenotypes resulting from lack of Ca²⁺/H⁺ exchange activity are due to altered Ca²⁺ transport or H⁺ transport.Open in a separate windowFigure 2Model of tonoplast Ca²⁺/H⁺ exchanger interaction with H⁺ pumps in response to salt stress. (A) In response to NaCl treatment, an elevation in cytosolic Ca²⁺ will occur, possibly due to vacuolar Ca²⁺ release.³ Increased CAX3-mediated Ca²⁺/H⁺ exchange activity¹⁴ will sequester excess Ca²⁺ into the vacuole. CAX3 may be involved in the generation of a specific Ca²⁺ signature that is recognised by the cell to mediate downstream stress responses. In addition, salt stress will lead to upregulation of H⁺ pumps at both the plasma membrane and the tonoplast (P-ATPase and V-ATPase)²⁵ which will in turn energize Na⁺/H⁺ exchange activity encoded by SOS1 and NHX1, promoting Na⁺ efflux from the cell. Increased V-ATPase activity may also upregulate Ca²⁺/H⁺ exchange. Activity of SOS1 requires activation by the kinase SOS2²⁴ which may also regulate tonoplast Na⁺/H⁺ exchange and V-ATPase activity.^23,24 (B) In a cax3 knockout mutant experiencing salt stress, the cytosolic Ca²⁺ elevation may be sustained due to reduced vacuolar Ca²⁺ sequestration and normal salinity-induced Ca²⁺ signalling pathways may be perturbed. Lack of CAX3 downregulates both P-ATPase and V-ATPase activity¹⁴ thereby reducing energization of the plasma membrane and tonoplast Na⁺/H⁺ exchangers and reducing Na⁺ efflux from the cell. Some energization of H⁺-coupled processes at the vacuole may be maintained by residual H⁺-pyrophosphatase (V-PPase) activity.The phenomenon observed between tonoplast Ca²⁺/H⁺ exchangers and H⁺ pumps at both the tonoplast and plasma membranes, suggesting a co-ordinate regulation between several transporters, is not solely restricted to this family of transporters. It is a common observation emerging from recent research on the manipulation of tonoplast transporters. Several labs have reported unpredictable phenotypes associated with ectopic expression of tonoplast proteins.^26–28 Until we understand the significance of these types of unexpected interactions, it is naïve to believe that engineering plants will provide predictable results.     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.