Ca2+-Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.     

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.     

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)- and Ca²⁺-mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H₂O₂-, and Ca²⁺-induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H₂O₂ compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca²⁺ inhibition of inward K⁺ currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Molecular and Physiological Analysis of Al3+ and H+ Rhizotoxicities at Moderately Acidic Conditions

Al³⁺ and H⁺ toxicities predicted to occur at moderately acidic conditions (pH [water] = 5–5.5) in low-Ca soils were characterized by the combined approaches of computational modeling of electrostatic interactions of ions at the root plasma membrane (PM) surface and molecular/physiological analyses in Arabidopsis (Arabidopsis thaliana). Root growth inhibition in known hypersensitive mutants was correlated with computed {Al³⁺} at the PM surface ({Al3+}_PM); inhibition was alleviated by increased Ca, which also reduced {Al3+}_PM and correlated with cellular Al responses based on expression analysis of genes that are markers for Al stress. The Al-inducible Al tolerance genes ALUMINUM-ACTIVATED MALATE TRANSPORTER1 and ALUMINUM SENSITIVE3 were induced by levels of {Al3+}_PM too low to inhibit root growth in tolerant genotypes, indicating that protective responses are triggered when {Al3+}_PM was below levels that can initiate injury. Modeling of the H⁺ sensitivity of the SENSITIVE TO PROTON RHIZOTOXICITY1 knockout mutant identified a Ca alleviation mechanism of H⁺ rhizotoxicity, possibly involving stabilization of the cell wall. The phosphatidate phosphohydrolase1 (pah1) pah2 double mutant showed enhanced Al susceptibility under low-P conditions, where greater levels of negatively charged phospholipids in the PM occur, which increases {Al3+}_PM through increased PM surface negativity compared with wild-type plants. Finally, we found that the nonalkalinizing Ca fertilizer gypsum improved the tolerance of the sensitive genotypes in moderately acidic soils. These findings fit our modeling predictions that root toxicity to Al³⁺ and H⁺ in moderately acidic soils involves interactions between both toxic ions in relation to Ca alleviation.Aluminum (Al), principally in the form of Al³⁺ released from soil clay minerals, is one of the most important rhizotoxic ions in acidic soils and is abundant in soil solutions at pH (water) of less than 5.0. Many forest and grass land species naturally adapted to acid soils are very tolerant of H⁺ and Al³⁺ and thrive in soils where the pH is less than 4.0. However, most crop plants used for agriculture show inhibitory growth effects, even when the soil pH is neutralized by liming to moderately acidic pH values in the range of pH 5 to 5.5. For example, crops sensitive to Al³⁺ and H⁺ such as turnip (Brassica rapa; Kinraide and Parker, 1990) and alfalfa (Medicago sativa; Yokota and Ojima, 1995) show growth inhibition at these soil pH values. Field research and soil experiments have shown that inhibitory effects of moderately acidic soils (pH > 5) can be ameliorated by the application of Ca fertilizers, even if they are nonalkalinizing, such as gypsum, and this leads to improvement in crop productivity (Carvalho and Van Raij, 1997; Mora et al., 1999). This indicates that the soil Ca status is an important factor in determining crop yield at moderately low soil pH values with regard to Al³⁺ and H⁺ rhizotoxicity occurring in these soils. An understanding of the complex situation of acid soil stress in soil pH in the range of pH (water) 5 to 5.5 is important for designing efficient soil acidity management and breeding programs for resistant crop use in low-input agricultural systems.The complex rhizotoxicities at moderately acidic conditions that can be alleviated by Ca have been predicted by modeling studies in wheat (Triticum aestivum; Kinraide, 2003). The model first computes the activity of the rhizotoxicants and alleviants at the plasma membrane (PM) surface, for example {Al3+}_PM and {H+}_PM, and {Ca2+}_PM, using a speciation-based Gouy-Chapman-Stern electrostatic (SGCS) model (Kinraide and Wang, 2010). The mechanisms of toxicity and alleviation are then modeled by regression analyses for root growth inhibition fitted to the nonlinear equations (Kinraide, 2003; Kinraide et al., 2004). The modeling studies well describe the complicated events in the Al-toxic solutions near the PM surface under moderately acidic conditions. For example, the elevation of pH from 4.5 to between 5 and 5.5 decreases the activity of the most rhizotoxic Al species, Al³⁺ in the solution ({Al3+}_bulk), while it increases the negativity of the PM surface because of dissociation of H⁺ from potentially negative ligands such as phospholipids. As a result, {Al3+}_PM remains at moderately high levels at the PM surface at pH greater than 5 due to the attraction to negative charges on the PM surface, but it can be alleviated by coexisting cations such as Ca²⁺ and even by another rhizotoxic cation, H⁺. These modeling studies have proposed different mechanisms of Ca alleviation in this complex situation (Kinraide, 1998; Kinraide et al., 2004). Mechanisms I (the electrostatic displacement of toxicant at the PM surface) and II (the restoration at the PM surface of Ca²⁺ electrostatically displaced by the toxicant) are events at the PM surface, but mechanism III, which explains the remaining portion of the Ca alleviation, may involve other physiological responses, including unknown mechanisms. These predictions, derived from the modeling study, likely explain the complex nature of moderately acidic soils but may require further validation because they were developed using root growth as the sole criterion for rhizotoxicity.Although these types of modeling approaches have not been performed using Arabidopsis (Arabidopsis thaliana) plants, clear symptoms of Al³⁺ and H⁺ rhizotoxicity at moderately acidic conditions (pH ≥ 5) has been identified in Arabidopsis (Kobayashi and Koyama, 2002; Iuchi et al., 2007). A quantitative trait locus study of Al tolerance at moderately acidic conditions (4 μm Al, pH 5; Kobayashi and Koyama, 2002) identified a very similar genetic architecture of Al tolerance to that derived from a study that employed a lower pH value but with a greater level of Al (50 μm Al, pH 4.2; Hoekenga et al., 2003). The former conditions employed a lower Ca concentration (200 μm) than the latter (3 mm), which accounted for the predictions of {Al3+}_PM in relation to {Ca2+}_PM by electrostatics. On the other hand, several Al³⁺- and H⁺-sensitive transfer DNA insertion knockout (KO) mutant genotypes have been identified using the lower ionic-strength moderately acidic media (Sawaki et al., 2009). These lines exhibit different degrees of hypersensitivity to moderately acidic conditions because of the dysfunction of different tolerance genes, suggesting the involvement of different mechanisms. In Arabidopsis, ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) regulates Al-activated root malate excretion that protects the root tip from acute Al toxicity by Al exclusion (Hoekenga et al., 2006), and ALUMINUM SENSITIVE3 (ALS3) regulates internal Al sequestration involved in long term Al tolerance (Larsen et al., 1997, 2005). The KO mutants for these genes display Al hypersensitivity. In addition, SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1)-KO, a suppressor of multiple genes for Al and H⁺ tolerance, shows sensitivity to Al³⁺ and H⁺ (Iuchi et al., 2007; Sawaki et al., 2009). These sensitive genotypes are useful tools for evaluating Al³⁺ and H⁺ toxicity in the pH range 5 to 5.5. On the other hand, several cellular responses, such as the induction of gene expression, have been identified in Arabidopsis that could be useful in the estimation of the attraction of {Al³⁺} to the PM, which is computed by our electrostatic-based model. Therefore, Arabidopsis appears to be a useful model system for the validation of modeling based on the SGCS model and to further our understanding of Al³⁺ and H⁺ rhizotoxicities at moderately acidic conditions in relation to Ca²⁺ alleviation.Computation of {Al3+}_PM requires accurate speciation of Al and other solutes in the bulk solution. The original SGCS program is suitable for relatively simple solutions (Kinraide and Wang, 2010). However, the rooting medium used for the Arabidopsis assays exceeds the number of solutes that can be accurately assessed by the SGCS program (Kobayashi et al., 2007). Consequently, we updated the modeling methodology using the speciation program GEOCHEM-EZ, which is suitable for complex media (Famoso et al., 2010; Shaff et al., 2010). This improved model, used in conjunction with molecular biological assays such as biomarker analysis of Al-inducible gene expression, has allowed us in this study to validate the predicted {Al3+}_PM rhizotoxicity in relation to {Ca2+}_PM alleviation from the wheat modeling studies. The updated modeling of Ca alleviation in mutants uncovered one of the mechanisms of Ca alleviation in the H⁺-sensitive mutant and identified an Al-sensitive double mutant genotype, phosphatidate phosphohydrolase1 (pah1) and pah2 (Nakamura et al., 2009), that fitted previous predictions. Finally, we demonstrate the ability of gypsum to ameliorate the sensitive phenotype of selected genotypes, when they were grown in moderately acidic soil culture. Taken together, we present here experimental validation of the SGCS-based modeling, and its combination with molecular physiology provides a deeper understanding of plant Al³⁺ and H⁺ toxicity in relation to Ca²⁺ alleviation at pΗ of at least 5.0.     

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).     

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.     

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.     

Distribution of Transglutaminase in Pear Pollen Tubes in Relation to Cytoskeleton and Membrane Dynamics

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca²⁺ dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.Transglutaminases (TGases [EC 2.3.2.13]; protein-Gln γ-glutamyltransferase) are a family of ubiquitous Ca²⁺-activated enzymes that are involved in animal cell morphogenesis and differentiation, apoptosis, cell death, inflammation, cell migration, and wound healing (Griffin et al., 2002; Mehta et al., 2006; Beninati et al., 2009). TGases are associated with different subcellular compartments, such as cytosol, plasma membrane, nucleus, mitochondria, and extracellular matrix. The specific localization of TGases is likely to determine both the biochemical activity and the type of proteins and/or substrates with which TGases react (Park et al., 2010). The distribution profile of TGase is affected by Ca²⁺, since the enzyme is preferentially associated with the lysosome compartment of liver cells in the absence of Ca²⁺ (Juprelle-Soret et al., 1984).TGase was initially detected in association with the cytosol, with the particulate (probably the microsomal) fraction (Birckbichler et al., 1976), and with the nucleus of animal cells (Remington and Russell, 1982). The association of TGase with the plasma membrane was related to its activity in promoting cell adhesion and to the interaction of cells with the extracellular matrix, while the presence of TGase in the nucleus is likely related to cell apoptosis (Griffin et al., 2002). How TGase is delivered to its final destination in animal cells remains to be clarified. Since the cytoskeleton is essential for the correct positioning of proteins in the cells, this interplay has often been studied in terms of potential substrates of TGase activity (Griffin et al., 2002). For example, the TGase-mediated incorporation of polyamines (PAs) stimulates actin polymerization (Takashi, 1988; Griffin et al., 2002). TGase was also found to associate with myosin in stress fibers of vascular smooth cells (Chowdhury et al., 1997). The association between TGase and microtubules (MTs) was initially studied in view of the importance of MTs in Alzheimer’s disease (Griffin et al., 2002), whereas the dynamics of MTs is also likely to be controlled by TGase (Al-Jallad et al., 2011). Interestingly, MTs are also a substrate of TGase activity in cells committed to apoptosis (Piredda et al., 1999). TGase was also shown to posttranslationally modify MT-associated proteins such as tau (Griffin et al., 2002).Information about the localization and function of TGases in plant cells is limited. Following the early evidence of an enzyme-based incorporation of PAs in plants (Serafini-Fracassini et al., 1988), a number of reports described the presence and role of TGase in nonphotosynthetic/photosynthetic tissues and in isolated chloroplasts (Serafini-Fracassini and Del Duca, 2008, and refs. therein). Attempts have also been made to examine the differences and similarities between plant and animal TGases. For example, a tobacco (Nicotiana tabacum) TGase was proposed to be involved in the programmed cell death (PCD) of the flower corolla (Della Mea et al., 2007); in such a case, TGase is likely to be released into the cell wall by a Golgi vesicle-based transport. Plant TGases might also be involved in protection against viruses (Del Duca et al., 2007) and in the self-incompatibility (SI) response involving pollen and stigma during sexual reproduction (Del Duca et al., 2010). Recently, different TGase isoforms were detected in meristematic apices of Jerusalem artichoke (Helianthus tuberosus) tuber sprouts (Beninati et al., 2013).The pollen tube is a widely investigated tip-growing plant cell (Lee and Yang, 2008). Studies are generally aimed at clarifying the many aspects related either to its growth or to rejection by the stigma/style. Early evidence for a role of PAs during pollen tube emergence (Bagni et al., 1981) was confirmed through the detection of PA binding via a Ca²⁺-activated TGase activity (Del Duca et al., 1997) and later by the identification of actin and tubulin as substrates of purified pollen TGase (Del Duca et al., 2009). In pollen, the enzyme affected the polymerization state and activity of actin filaments (AFs) and MTs (Del Duca et al., 2009) and existed as both soluble and cell wall associated (Di Sandro et al., 2010). Visualization of fluorescently labeled TGase products indicated that the cross-linking activity of TGase occurred at the apex of pollen tubes, in a basal region close to the pollen grain and within the pollen grain itself (Iorio et al., 2008). The enzyme was found as a soluble cytoplasmic form likely involved in the regulation of unspecified physiological processes (possibly associated with the cytoskeleton; Del Duca et al., 2009).Although the association of pollen TGases with organelles/vesicles has not been reported, an extracellular form of a Ca²⁺-dependent TGase was shown to be involved in pollen tube growth (likely as a modulator of cell wall building and strengthening). Moreover, pollen TGase was secreted in the incubation medium during germination, where it might catalyze the cross linking of PAs with secreted proteins (Di Sandro et al., 2010). This suggests that pollen TGase may be secreted through a vesicle-based mechanism. Finally, a TGase activity was also observed in planta, consistent with a possible role of TGase during tube migration through the style (Di Sandro et al., 2010) or in the SI response of pollen tubes (Del Duca et al., 2010).The pollen tube is an excellent model to study how a given plant protein is either secreted or delivered to its final destination. Although we know that actin and tubulin are substrates of TGase activity, and that the active enzyme is located in the cell wall and released outside, how TGase is distributed in the cells and how this process is dependent on cytoskeleton and membrane dynamics remain unknown. Here, we wanted to study in detail the localization and distribution of TGase in growing pollen tubes of pear (Pyrus communis) in relation to both cytoskeleton and membrane dynamics. The aim was to shed light on the mechanism by which TGase is transported and secreted, a process that is still not well understood even in animal cells. Specific antibodies that cross react with the TGase of pollen tubes were used to localize the enzyme in different membrane compartments and in the cell wall. The use of specific inhibitors indicated that the delivery of extracellular TGase is dependent on both AFs and membrane dynamics. Analysis by bidimensional electrophoresis (2-DE) showed that distinct TGase isoforms are associated with different cell compartments, suggesting that TGase might be differently regulated according to its position in the cell. Together, these data may contribute to our understanding of the mechanisms underlying pollen tube growth, an essential aspect of fertilization processes.     

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells

Oscillations in cytosolic-free Ca²⁺ concentration ([Ca2+]_i) have been proposed to encode information that controls stomatal closure. [Ca2+]_i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca2+]_i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca2+]_i and, with the accompanying oscillations in voltage, drive the K⁺ and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.Stomata in the leaf epidermis are the main pathway both for CO₂ entry for photosynthesis and for foliar water loss by transpiration. Guard cells surround the stomatal pore and regulate the aperture, balancing the often conflicting demands for CO₂ and water conservation. Guard cells open and close the pore by expanding and contracting through the uptake and loss, respectively, of osmotic solutes, notably of K⁺, Cl⁻, and malate²⁻ (Mal²⁻; Pandey et al., 2007; Kim et al., 2010; Roelfsema and Hedrich, 2010; Lawson and Blatt, 2014). These transport processes comprise the final effectors of a regulatory network that coordinates transport across the plasma membrane and tonoplast, and maintains the homeostasis of the guard cell. A number of well-defined signals—including light, CO₂, drought and the water stress hormone abscisic acid (ABA)—act on this network, altering transport, solute content, turgor and cell volume, and ultimately stomatal aperture.Much research has focused on stomatal closure, underscoring both Ca²⁺-independent and Ca²⁺-dependent signaling. Of the latter, elevated cytosolic-free Ca²⁺ concentration ([Ca2+]_i) inactivates inward-rectifying K⁺ channels (I_K,in) to prevent K⁺ uptake and activates Cl⁻ (anion) channels (I_Cl) at the plasma membrane to depolarize the membrane and engage K⁺ efflux through outward-rectifying K⁺ channels (I_K,out; Keller et al., 1989; Blatt et al., 1990; Thiel et al., 1992; Lemtiri-Chlieh and MacRobbie, 1994). ABA, and most likely CO₂ (Kim et al., 2010), elevate [Ca2+]_i by facilitating Ca²⁺ entry at the plasma membrane to trigger Ca²⁺ release from endomembrane stores, a process often described as Ca²⁺-induced Ca²⁺ release (Grabov and Blatt, 1998, 1999). The hormone promotes Ca²⁺ influx by activating Ca²⁺ channels (I_Ca) at the plasma membrane, even in isolated membrane patches (Hamilton et al., 2000, 2001), which is linked to reactive oxygen species (Kwak et al., 2003; Wang et al., 2013). In parallel, cADP-ribose and nitric oxide promote endomembrane Ca²⁺ release and [Ca2+]_i elevation (Leckie et al., 1998; Neill et al., 2002; Garcia-Mata et al., 2003; Blatt et al., 2007). Best estimates indicate that endomembrane release accounts for more than 95% of the Ca²⁺ entering the cytosol to raise [Ca2+]_i (Chen et al., 2012; Wang et al., 2012).One feature of stomatal response to ABA, and indeed to a range of stimuli both hormonal as well as external, is its capacity for oscillations both in membrane voltage and [Ca2+]_i. Guard cell [Ca2+]_i at rest is typically around 100 to 200 nm, as it is in virtually all living cells. In response to ABA, [Ca2+]_i can rise above 1 μm—and locally, most likely above 10 μm—often in cyclic transients of tens of seconds to several minutes’ duration in association with oscillations in voltage and stomatal closure (Gradmann et al., 1993; McAinsh et al., 1995; Webb et al., 1996; Grabov and Blatt, 1998, 1999; Staxen et al., 1999; Allen et al., 2001). In principle, cycling in voltage and [Ca2+]_i arises as closure is accelerated with a controlled release of K⁺, Cl⁻, and Mal²⁻ from the guard cell and is subject to extracellular ion concentrations (Gradmann et al., 1993; Chen et al., 2012). However, it has been proposed that these, and similar oscillations in a variety of plant cell models, serve as physiological signals in their own right (McAinsh et al., 1995; Ehrhardt et al., 1996; Taylor et al., 1996). In support of such a signaling role, experiments designed to impose [Ca2+]_i (and voltage) oscillations in guard cells have yielded an optimal frequency for closure with a period near 10 min (Allen et al., 2001). Nonetheless, the studies offer no mechanistic explanation for this optimum that could validate a causal role in signaling, and none has been forthcoming since. Here we address questions of how such optimal frequencies in [Ca2+]_i oscillation arise and their relevance for stomatal closure, using quantitative systems analysis of guard cell transport and homeostasis. Our findings indicate that oscillations in voltage and [Ca2+]_i, and their optima associated with stomatal closure, are most simply explained as emerging from the interactions between ion transporters that drive stomatal closure. Thus, we conclude that these oscillations do not control, but are a by-product of the transport that determines stomatal aperture.     

The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific,Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.     

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca²⁺ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth.