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ScienceDirect Signaling in cells and organisms — calcium holds the line Leonie Steinhorst and Jo¨rg Kudla Previous research has established calcium (Ca2+) and reactive oxygen species (ROS) as important cellular second messengers in eukaryotes. Recently, the occurrence of cell-tocell moving Ca2+ and ROS waves was reported in plants. This was paralleled by the discovery of long-distance woundactivated surface potential changes (WASPs) that require the function of putatively Ca2+-releasing glutamate receptor-like channels (GLRs) in Arabidopsis. Although the functional interconnection of Ca2+-dependent phosphorylation and ROS waves via NADPH oxidase activation has been clearly established, potential further interconnections between these long-distance signaling processes are less clear. In this review we cover emerging concepts and existing open questions that interconnect cellular and global signaling via Ca2+, ROS and WASPs. Addresses Institut fu¨r Biologie und Biotechnologie der Pflanzen, Universita¨t Mu¨nster, Schlossplatz 4, 48149 Mu¨nster, Germany Corresponding author: Kudla, Jo¨rg ([email protected])

Current Opinion in Plant Biology 2014, 22:14–21 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´

http://dx.doi.org/10.1016/j.pbi.2014.08.003 1369-5266/# 2014 Elsevier Ltd. All right reserved.

Introduction 2+

Calcium ions (Ca ) function as versatile and important second messengers in animals and plants [1–7]. During signaling processes, temporally and spatially defined changes in cytoplasmic Ca2+ concentration that occur in response to specific stimuli represent signal information to the cell and the organism. Since the shape, the intensity and the duration of these cytoplasmic Ca2+ elevations are often specifically defined by the nature of the inducing stimulus these elevations have been designated as Ca2+ signatures [8,9]. Cytoplasmic Ca2+ signatures can be generated by release from internal Ca2+ stores, like the vacuole, and influx from the outside. The formation of a temporally defined Ca2+ signal requires tightly regulated and coordinated processes of Ca2+-influx into the cytosol and subsequent removal back Current Opinion in Plant Biology 2014, 22:14–21

into internal stores or the apoplast. In non-induced cells the resting cytoplasmic Ca2+ concentration is usually kept at concentrations of around 100–200 nM, while the concentration in the storage compartments (like the vacuole and the apoplast) can easily reach up to 10 mM [10]. This steep gradient easily facilitates the generation of transient cytoplasmic Ca2+ signatures that can reach up to several hundred micromolar concentrations at their maximum. While classical animal Ca2+ release channels like the IP3 receptors and the STIM/ORAI system appear to be absent in higher plants, emerging evidence points to a crucial role of cation channels like glutamate receptorlike (GLR) and cyclic nucleotide-gated channels (CNGCs) in the formation of Ca2+ signals in plants [11,12].. A second important level of Ca2+ signaling is the translation of the Ca2+ signature into specific cellular response reactions. This is brought about by Ca2+-binding proteins that function as Ca2+ sensors. Upon Ca2+-binding that adds negative charges to these proteins they undergo conformational changes. Ca2+ sensors that combine an enzymatic domain (like a kinase domain) and a Ca2+-binding domain in one protein can translate a Ca2+ signature in a monomolecular reaction into response reactions (like phosphorylation of substrates) and are designated as sensor-responders [4]. In contrast, Ca2+ sensors that lack an enzymatic domain are designated as sensor-relays. These proteins will upon Ca2+-binding change their conformation, interact with target proteins and thereby convey the Ca2+ signal in a bi-molecular reaction. These target proteins can be kinases which upon interaction with Ca2+-binding proteins undergo activation. Plants are equipped with an elaborate toolkit of such Ca2+ sensors. These involve the large gene-family of Ca2+dependent protein kinases (CDPKs) that comprises 34 members in Arabidopsis that function as sensor-responders [13]. Calmodulin proteins (CaMs, four members in Arabidopsis) and calmodulin-like proteins (CMLs, around 50 members in Arabidopsis) function as sensor-relays [14]. In addition, calcineurin B-like proteins (CBLs, 10 members in Arabidopsis) can convey the Ca2+ signal by interaction with CBL-interacting protein kinases (CIPKs, 26 members in Arabidopsis) [15]. The existence of such large Ca2+ decoding gene families in plants suggested their diverse and important roles in many different response reactions and physiological processes. And indeed, recent studies have unraveled the function of CDPKs and CBL-CIPK complexes in various processes like www.sciencedirect.com

Signaling in cells and organisms Steinhorst and Kudla 15

pollen tube growth, guard cell regulation, plant morphogenesis, abiotic stress responses and pathogen responses [16,17–25]. Also on the cellular level our understanding concerning the function of these Ca2+ decoding kinases has greatly advanced by the identification of ion channels, transcription factors and NADPH oxidases as their target proteins [26,27–33]. An exciting new development during especially the past two years is the emerging new concept that Ca2+ signals and Ca2+-dependent phosphorylation in connection with the defined transient formation of reactive oxygen species (ROS) signals contribute to long-distance signaling and intercellular response coordination in plants (Figure 1a). Moreover, in several biological processes that require long-distance signaling both second messengers appear to be somehow interlaced with the reiterative generation of electrical signals and hormonal responses [21,34–36,37] (Figure 1d). In the following we will focus on new findings that influence the emerging concept of multiple messenger long-distance signaling processes. We will put special emphasis on discussing emerging consequences and open questions.

Ion channels that function in Ca2+ signaling A long-lasting and fundamental conundrum in plant research is about the identity of the ion channels that underlie the generation of cytoplasmic Ca2+ signals. Such channels should be found at the plasma membrane but also at internal membranes like the tonoplast or the endoplasmic reticulum membrane. It is by now generally accepted that in higher plants true homologs of classical Ca2+-specific ion channels like IP3 receptors and voltagegated Ca2+ channels (like CaVs) are absent, while they have been identified in the genomes of several algae [11,38]. Also the STIM/ORAI Ca2+ channel that functions in Ca2+-induced Ca2+ release (CICR) in metazoans is not present in higher plants [39]. This supports the notion that plants have evolved specific Ca2+ release systems. However, it needs to be considered that clear electrophysiological evidence supports the existence of Ca2+specific voltage-gated and mechano-activated channels in plants, despite our lack of knowledge about their molecular identity [4,40]. However, for several members of the family of CNGCs in Arabidopsis it has become increasingly evident that they contribute to Ca2+ signaling in plants. CNGCs are nonspecific cation channels that form a family of 20 members in Arabidopsis and are regulated by cyclic nucleotides [41–43]. CNGCs have been shown to function in the context of developmental processes, abiotic stress responses, the establishment of thermo-tolerance and the regulation of pollen tube tip growth [44–48]. The first evidence that linked CNGC function to Ca2+-dependent processes was gained by analyses that revealed a functional requirement of CNGC18 for pollen tube tip growth and male fertility [44]. Importantly, a recent study www.sciencedirect.com

provided strong evidence that CNGC18 functions as Ca2+-permeable divalent cation-selective channel in HEK293T cells that is activated by cGMP and cAMP [49]. Quite remarkably, most recently it was reported that the CDPK CPK32 interacts with and activates CNGC18 [50]. These conclusions were based on electrophysiological studies in Xenopus oocytes and transient overexpression studies in pollen tubes. Together these findings provide strong support for a role of CNGC18 in Ca2+ entry into the cytoplasm via the plasma membrane. In this regard it will be most interesting to perform similar studies for additional members of the CNGC family. Importantly, the latter publication also points to a Ca2+-dependent regulation of Ca2+-influx by Ca2+-dependent kinases. This phenomenon may provide an important mechanism for feedback regulation in Ca2+ signaling. The GLR family in Arabidopsis encompasses 20 members that are involved in diverse biological processes like abiotic stress responses, plant pathogen interactions, root morphogenesis and pollen tube growth [51–58]. Recently, the importance of GLRs for the generation of Ca2+ currents was established for GLR1.2 [59]. Mutant analyses revealed that this channel is required for proper Ca2+-fluxes underlying pollen tube growth and fertility. Ca2+ conductance of pollen GLRs has been established with Ca2+-specific vibrating probe measurements that involved GLR agonists (D-serine) or antagonists. Importantly, a complementary study that electrophysiologically characterized GLR3.4 in HEK cells characterized this GLR as an amino acid gated channel that was suggested to be highly selective for Ca2+ and capable of inducing cytoplasmic Ca2+ signatures in response to Asn, Gly and Ser [60]. In addition, two independent studies of GLR 3.3 provided evidence that this GLR has a crucial role as a Ca2+ channel in plant defense [61,62]. Although animal Glu receptors have been reported to be subject to regulation by phosphorylation that modulates receptor trafficking and channel properties this potential aspect of GLR regulation has not been reported in plants. Considering the intricate interconnection of plant GLRs (see below) with long-distance signaling processes in plants their regulation represents an important question to be addressed in the future. A breakthrough study by E. Farmer and colleagues in 2013 established a crucial role of several GLRs in longdistance signaling in response to wounding [37]. In response to wounding — as it occurs naturally upon herbivore attack to plants — a systemic long-distance signal is induced that informs distant parts of the plants about the encountered attack and results in distal production of jasmonates — plant hormones that enhance defense reactions. In this work the authors used noninvasive electrodes to map wound-activated surface potential changes (WASPs) in Arabidopsis thaliana after Current Opinion in Plant Biology 2014, 22:14–21

16 Cell biology

Figure 1

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Models of cell-to-cell signal propagation in response to local stimuli in Arabidopsis. (a) Apoplastic ROS generated by RBOHD mediate signal propagation in long-distance signaling in response to diverse stimuli [76]. RBOHD is phospho-regulated by the Ca2+-regulated kinase CPK5 and probably also by CIPKs [16] (Ko¨ster and Kudla, unpublished). (b) In response to a local salt-stress a Ca2+ wave travels from cell-to-cell. The speed of this Ca2+ wave depends on the activity of the vacuolar ion channel TPC1 and might be associated with a ROS signal [63]. (c) Model of woundactivated surface potential changes (WASPs) propagating from cell-to-cell [37]. The propagation of these electrical signals is controlled by GLRs that are thought to conduct Ca2+ across the plasma membrane, thereby generating an intracellular Ca2+ signal. (d) Hypothetical model of signal propagation combining the models depicted in a–c.

Current Opinion in Plant Biology 2014, 22:14–21

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Signaling in cells and organisms Steinhorst and Kudla 17

wounding of a specific leaf. This approach revealed that electrical signals, propagated likely predominantly via the vascular system with a speed of 2–9 cm/min, and elicited jasmonate biosynthesis resulting in induction of jasmonate-induced genes in distal leaves. Importantly, this work identified the glutamate receptor-like ion channels GLR3.2, 3.3 and 3.6 as crucial components of systemic signaling since mutations in these genes attenuated WASPs. Moreover, a glr3.3glr3.6 double mutant displayed reduced distal jasmonate responses. Together, these findings established a role of Ca2+ release channels in plant long-distance signaling and linked them to the propagation of electrical signals (Figure 1c). The role of such Ca2+ signals in long-distance signaling in plants was excitingly illuminated in a recent work by S. Gilroy and colleagues [63]. The authors studied Arabidopsis plants that expressed a highly sensitive genetically encoded Nano-YC Ca2+ reporter protein. Upon local application of NaCl stress they not only observed a local induction of a Ca2+ signature, but also Ca2+ waves originating at the site of application and propagating through the plant with a speed of approximately 2–3 cm/min (Figure 1b). Such Ca2+ waves were not observed in response to cold, touch and osmotic stress under these experimental conditions. However, earlier studies using the Ca2+ reporter protein aequorin reported the occurrence of wave-like propagating Ca2+ signals in leaves of tobacco that were locally challenged with wounding or cold-shocks [64]. Importantly, an independent study by Xiong and colleagues that used a BRET-based Ca2+ indicator to study Ca2+ waves in leaves reported a similar speed of up to 3 cm/min [65]. The comprehensive study by Choi et al. also addressed rapid and long-term salt induced changes in gene expression in distant tissues. This identified the gene for the vacuolar Two-Pore Channel 1 (TPC1) as fast and strongly upregulated. Remarkably, mutation of TPC1 resulted in a 25-fold slowing of the salt-induced Ca2+ wave and overexpression of TPC1 increased the speed of the Ca2+ wave by 1.7-fold. TPC1 encodes for the slow vacuolar (SV) channel and tpc1 mutant plants lacked the slow vacuolar channel activity and were defective in guard cell responses to extracellular Ca2+ [66]. Because of its complex regulation and its low selectivity for Ca2+ ions the involvement of TPC1 in the formation of Ca2+ signals is poorly understood and controversially discussed [67–70]. However, the prominent role of this channel for the formation of Ca2+ waves strongly argues for further studies on this channel. Interestingly, in earlier studies a constitutively active mutant of TPC1 ( fou2) was found to exhibit increased jasmonate levels in response to wounding, presumably evoked by enhanced vacuole-derived cytosolic Ca2+ signals [71–73]. These findings might point to a connection to GLR-mediated jasmonate signaling and WASPs and deserve further investigation. www.sciencedirect.com

Interconnection of Ca2+ waves and ROS signaling Similar to Ca2+, different ROS species have been established as important second messengers in cellular signaling in plants [12,74,75]. This concept was significantly advanced in 2009 when R. Mittler and colleagues reported the occurrence of ROS waves that traveled long distances in Arabidopsis [76]. The authors took advantage of transgenic Arabidopsis lines that expressed a luciferase reporter protein under control of the H2O2-induced ZAT12 promoter to visualize ROS production in plants. When they locally wounded Arabidopsis plants they observed ROS waves that propagated through the tissues with a speed of up to 8.4 cm/min. Genetic analyses identified a crucial role for the NADPH oxidase ‘respiratory burst oxidase homolog D’ (RBOHD) in these processes. In rbohd mutant plants the speed and intensity of ROS waves were dramatically reduced but not fully abolished. In subsequent studies the rapid systemic ROS signal was also triggered in response to heat, cold, high-intensity light and salinity stress [76]. These results established a profound role of ROS signals in mediating rapid cell-to-cell propagating long-distance signals in plants. A most recent study on the regulation of the NADPH oxidase RBOHD during rapid defense signal propagation provided a most exciting interconnection of Ca2+-dependent protein phosphorylation, NADPH oxidase mediated ROS waves and defense long-distance signaling [16]. In this study the authors identified the Ca2+ regulated kinase CPK5 as being rapidly activated in response to pathogen-associated molecular pattern (PAMP) stimulation. Selected reaction monitoring MS identified the plant NADPH oxidase RBOHD as an in vivo phosphorylation target of CPK5. Importantly, CPK5dependent in vivo phosphorylation of RBOHD was induced by both PAMP and ROS stimulation. Moreover, rapid CPK5-dependent activation of defense reactions at sites distal to the infection site was compromised in cpk5 and rbohd loss-of-function mutants. Together these data support a model in which CPK5 and RBOHD are key components of a self-propagating activation circuit mediating cell-to-cell communication during pathogen responses (Figure 1a). Another recent study reported that the closely related NADPH oxidase RBOHF which often functions together with RBOHD [77,78] is subject to regulation by a different family of Ca2+ regulated kinases [26]. Here it was shown that the Ca2+ sensors CBL1 and CBL9 together with their interacting kinase CIPK26 activate the ROS generation by RBOHF in a Ca2+-dependent manner. Moreover, CIPK26 appears also to be able to activate not only RBOHF but also RBOHD (Ko¨ster and Kudla, unpublished). These data suggest a much more complex interconnection of the Ca2+-decoding CDPK and CBL-CIPK families with the family of plant NADPH oxidases that has 10 members in Arabidopsis [79]. Current Opinion in Plant Biology 2014, 22:14–21

18 Cell biology

Do plants have a unifying long-distance signaling system? In animals it is clear that long-distance communication is brought about by a complex and differentiated nervous system that combines at different steps the function of electrical signals, protein phosphorylation and second messengers like Ca2+ and ROS. Also in plants it is evident that for example a salt-stress signal that is primarily perceived at the root needs to trigger adaptive responses in the aerial parts of the plant. Similarly, systemic acquired resistance involves communication between locally challenged and distal leaves. Plants obviously had to evolve their own solutions to deal with such challenges. The discussed examples of WASPs, ROS and Ca2+ waves as well as the identification of GLRs, TPC1, RBOHD and CPK5 as important components in different long-distance signaling processes provide first important insights into how plants solved these problems [16,37,63,76]. Best understood at present appears to be the interconnection of Ca2+-dependent phosphorylation via CDPKs (and likely CBL-CIPKs) with the formation and propagation of a ROS wave. In this mechanism a stimulus (like wounding) initiates a rise in intracellular Ca2+, causing activation of CPK5 (and potentially other CPKs and CBL-CIPKs, especially in response to different stimuli) that in turn phosphorylation-dependently activates RBOHD [16]. Extracellular ROS generated by this NADPH oxidase represents the cell permeable signal to neighboring cells where perception of H2O2 induces further Ca2+ release and thereby reiterations of Ca2+-dependent CPK5 activation and RBOHD phosphorylation, resulting in a Ca2+/ mediated signaling relay phosphorylation/ROS (Figure 1a,d). Obviously it will be most interesting to elucidate if similar mechanisms function in other plant signaling processes, for example responses to abiotic stresses. However, the history of these findings also highlights our current limitations in cross-comparing the different experiments. This comes in part from the different sensitivity of the reporter systems and assays that are combined to investigate long-distance signaling. For example in the initial report on the formation of the ROS wave the application of the Ca2+ channel blocker LaCl3 did not abolish the long-distance propagating ROS wave, arguing against a role of Ca2+ in this process [76]. Remarkably, even at this time the authors carefully discussed that they could not exclude the possibility that LaCl3 did not penetrate the tissue efficiently enough to prevent the ROS wave. Also Choi et al. reported that LaCl3 inhibited the Ca2+ wave but did not block the upregulation of saltinduced genes [63]. In addition, Mousavi et al. observed that DPI, an inhibitor of the ROS wave, reduced WASP duration but did not affect wound-induced gene expression [37]. Also the rbohd mutant that has been shown to Current Opinion in Plant Biology 2014, 22:14–21

reduce and delay the ROS wave did not affect woundinduced gene expression. These findings could support the conclusion that the Ca2+ wave is not required for regulating salt-induced gene expression and a ROS wave is not required for wound-induced gene expression. However, if we consider the limited sensitivity of our reporter proteins and assays it appears conceivable that a strongly reduced intensity of a propagating signal (that for example cannot be visualized by the YCNano Ca2+ reporter protein or a Zat12::Luciferase ROS reporter system) may still occur below the detection threshold, while the highly sensitive qRT-PCRs used to analyze gene expression could still detect responses on this level. Similarly, a decelerated propagating signal — as was observed in rbohd, tpc1 and glr mutants — could still trigger a transcriptional response even if it arrives later (or weaker) at the distal part of a plant. A (currently hypothetical) ideal solution to this problem could lie in the simultaneous expression of cell-biological reporter proteins for Ca2+, ROS and electrical potentials and their simultaneous analysis during one defined stimulusresponse in a single plant. To this end one could for example use the sensitive R-GECO Ca2+ reporter proteins that visualize Ca2+ dynamics in the red spectrum [80] with the ratiometric H2O2 reporter protein HyPer that visualizes ROS dynamics in the yellow spectrum [81]. Moreover, a ratiometric protein for visualizing membrane potential changes (voltage-sensitive fluorescent protein; VSFP) has been recently reported [82]. Successfully combining such tools would allow a new quality of studies on plant (long-distance) signaling. The currently reported speeds of signal propagation with up to 8.4 cm/min for ROS, 2–3 cm/min for Ca2+ and 2–9 cm/min for WASP signal waves are of similar magnitude and would allow for a combined function of these messengers [37,63,65,76]. Currently existing differences may again be a result of the different sensitivities or reaction kinetics of the applied reporter systems. In addition, it needs to be considered that the pace of ROS waves was calculated based on data that were obtained with a transcriptional reporter system. Taking into account, that the transcription/translation of the luciferase reporter requires some time, this currently leaves some uncertainty about the current speed calculations. More complicated appears to unite the different stimuli that caused or did not cause the respective response waves. Here the situation is most simple for the ROS wave because it has been observed in response to many different stimuli such as wounding, heat, cold, salt and high light [76]. However, Choi et al. observed that many abiotic stresses induced a local Ca2+ response, but that only salt-stress triggered a long-distance propagating Ca2+ wave [63]. Propagating Ca2+ signals have also been previously observed in response to cold and wounding in tobacco leaves using a different reporter protein [64]. Therefore it is tempting to speculate that www.sciencedirect.com

Signaling in cells and organisms Steinhorst and Kudla 19

the use of more sensitive Ca2+ reporters like R-GECO will allow identifying of Ca2+ waves in response to additional stimuli. Currently the role of WASPs and GLRs in long-distance signaling has been detailed only in response to wounding but plasma membrane depolarizations are common in plants [37]. In any case it will be most interesting to determine in more detail which array of stimuli induces which types of long-distance signals and how stimulus specificity in this process is achieved. Such advancements could involve the integration of additional signal types like pressure changes. In this regard, for wound responses most recently an interesting squeeze cell hypothesis was formulated [83]. This hypothesis predicts that xylem-transmitted pressure changes in wounded plants provide a rapid long-distance distal signaling mechanism that could be converted into a further radial propagated response in xylem contact cells by Ca2+ release via GLRs. Such a mechanism could also elegantly interconnect very rapid pressure signals with the Ca2+/ROS waves signal propagating system. As usual, exciting findings raise important questions. For example, is GLR function also required for the formation of Ca2+ and ROS waves? The inhibitor studies by Choi et al. indicate that plasma membrane channels as well as endogenous Ca2+ channels contribute to the formation of the Ca2+ wave [63]. GLRs that are related to GLR3.3 and GLR3.6 which function in wound responses could fulfill such a function. Is there a role for Ca2+-dependent phosphorylation by CDPKs and CBL-CIPKs in regulating Ca2+ waves and WASPs? Do the same isoforms of the identified NADPH oxidases and GLRs function in different long-distance signaling waves, or contribute RBOHD only to ROS waves and GLR3.3 and GLR3.6 only to WASPs? Similarly, would different stimuli involve the function of distinct NADPH oxidases, GLRs and Ca2+ regulated kinases? In this regard it is tempting to speculate that Ca2+-dependent phosphorylation may represent a means of coordinating the activity of GLRs, NADPH oxidases and TPC1. Clearly, we are in the beginning of an exciting period in advancing our understanding of this fundamental phenomenon of plant signaling.

Acknowledgements The work in the author’s laboratory was supported by grants from the DFG (SFB629 and FOR964), BMBF and DAAD.

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10. Bush DS: Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 1995, 46:95-122. 11. Wheeler GL, Brownlee C: Ca2+ signalling in plants and green algae–changing channels. Trends Plant Sci 2008, 13:506-514. 12. Steinhorst L, Kudla J: Calcium and reactive oxygen species rule the waves of signaling. Plant Physiol 2013, 163:471-485. 13. Harper JF, Breton G, Harmon A: Decoding Ca2+ signals through plant protein kinases. Annu Rev Plant Biol 2004, 55:263-288. 14. Batisticˇ O, Kudla J: Calcium: not just another ion. In Cell Biology of Metals and Nutrients. Edited by Hell R, Mendel RR. SpringerVerlag; 2010:17-54. 15. Weinl S, Kudla J: The CBL-CIPK Ca2+-decoding signaling network: function and perspectives. New Phytol 2009, 184:517-528. 16. Dubiella U, Seybold H, Durian G, Komander E, Lassig R, Witte CP,  Schulze WX, Romeis T: Calcium-dependent protein kinase/ NADPH oxidase activation circuit is required for rapid defense signal propagation. Proc Natl Acad Sci U S A 2013, 110:8744-8749. This paper is of fundamental importance because it advances the general concepts of signal transduction in plants. The authors report a crucial function of the Ca2+-regulated kinase CPK5 in plant pathogen responses and identify a NADPH oxidase as target of this kinase. Importantly, this work advances existing long-distance signaling models that involve the propagation of ROS waves by reporting a self-propagating activation circuit in which CPK5 and the NADPH oxidase RBOHD facilitate rapid long-distance signal propagation in defense signaling. 17. Ma¨hs A, Steinhorst L, Han J, Shen L, Wang Y, Kudla J: The calcineurin B-like Ca2+ sensors CBL1 and CBL9 function in pollen germination and pollen tube growth in Arabidopsis. Mol Plant 2013, 6:1149-1162. 18. Gutermuth T, Lassig R, Portes MT, Maierhofer T, Romeis T, Borst JW, Hedrich R, Feijo JA, Konrad KR: Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20. Plant Cell 2013, 25:4525-4543. 19. Lassig R, Gutermuth T, Bey TD, Konrad KR, Romeis T: Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth. Plant J 2014, 78:94-106. 20. Matschi S, Werner S, Schulze WX, Legen J, Hilger HH, Romeis T: Function of calcium-dependent protein kinase CPK28 of Arabidopsis thaliana in plant stem elongation and vascular development. Plant J 2013, 73:883-896. 21. Romeis T, Herde M: From local to global: CDPKs in systemic defense signaling upon microbial and herbivore attack. Curr Opin Plant Biol 2014, 20C:1-10. 22. Mori IC, Murata Y, Yang Y, Munemasa S, Wang YF, Andreoli S, Tiriac H, Alonso JM, Harper JF, Ecker JR et al.: CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anionand Ca2+-permeable channels and stomatal closure. PLoS Biol 2006, 4:e327. Current Opinion in Plant Biology 2014, 22:14–21

20 Cell biology

23. D’Angelo C, Weinl S, Batisticˇ O, Pandey GK, Cheong YH, Schu¨ltke S, Albrecht V, Ehlert B, Schulz B, Harter K et al.: Alternative complex formation of the Ca2+-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis. Plant J 2006, 48:857-872. 24. Cheong YH, Pandey GK, Grant JJ, Batisticˇ O, Li L, Kim BG, Lee SC, Kudla J, Luan S: Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis. Plant J 2007, 52:223-239. 25. Franz S, Ehlert B, Liese A, Kurth J, Cazale AC, Romeis T: Calciumdependent protein kinase CPK21 functions in abiotic stress response in Arabidopsis thaliana. Mol Plant 2011, 4:83-96. 26. Drerup MM, Schlu¨cking K, Hashimoto K, Manishankar P,  Steinhorst L, Kuchitsu K, Kudla J: The calcineurin B-like calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26 regulate the Arabidopsis NADPH oxidase RBOHF. Mol Plant 2013, 6:559-569. This work establishes that not only CDPK proteins but also CBL Ca2+ sensors, together with their interacting CIPKs, bring about Ca2+-dependent activation of NADPH oxidases. 27. Held K, Pascaud F, Eckert C, Gajdanowicz P, Hashimoto K, Corratge-Faillie C, Offenborn JN, Lacombe B, Dreyer I, Thibaud JB, Kudla J: Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex. Cell Res 2011, 21:1116-1130. 28. Kudla J, Batisticˇ O, Hashimoto K: Calcium signals: the lead currency of plant information processing. Plant Cell 2010, 22:541-563. 29. Geiger D, Maierhofer T, Al-Rasheid KA, Scherzer S, Mumm P, Liese A, Ache P, Wellmann C, Marten I, Grill E et al.: Stomatal closure by fast abscisic acid signaling is mediated by the guard cell anion channel SLAH3 and the receptor RCAR1. Sci Signal 2011, 4:ra32. 30. Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, Al-Rasheid KA, Grill E et al.: Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities. Proc Natl Acad Sci U S A 2010, 107:8023-8028. 31. Boudsocq M, Sheen J: CDPKs in immune and stress signaling. Trends Plant Sci 2013, 18:30-40. 32. Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M, Shimamoto K, Doke N, Yoshioka H: Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 2007, 19:1065-1080. 33. Asai S, Ichikawa T, Nomura H, Kobayashi M, Kamiyoshihara Y, Mori H, Kadota Y, Zipfel C, Jones JD, Yoshioka H: The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase. J Biol Chem 2013, 288:14332-14340. 34. Suzuki N, Mittler R: Reactive oxygen species-dependent wound responses in animals and plants. Free Radic Biol Med 2012, 53:2269-2276. 35. Suzuki N, Miller G, Salazar C, Mondal HA, Shulaev E, Cortes DF, Shuman JL, Luo X, Shah J, Schlauch K et al.: Temporal-spatial interaction between reactive oxygen species and abscisic acid regulates rapid systemic acclimation in plants. Plant Cell 2013, 25:3553-3569. 36. Baxter A, Mittler R, Suzuki N: ROS as key players in plant stress signalling. J Exp Bot 2014, 65:1229-1240. 37. Mousavi SA, Chauvin A, Pascaud F, Kellenberger S, Farmer EE:  GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling. Nature 2013, 500:422-426. This paper is of eminent importance because it advances our understanding of long-distance signal transduction in plants. The authors report that wounding of specific leaves induces an electrical signal that propagates through the plant to distal leaves and they provide strong evidence that specific Ca2+-releasing glutamate receptor-like ion channels (GLRs) are functionally required for long-distance signaling. Current Opinion in Plant Biology 2014, 22:14–21

38. Nagata T, Iizumi S, Satoh K, Ooka H, Kawai J, Carninci P, Hayashizaki Y, Otomo Y, Murakami K, Matsubara K, Kikuchi S: Comparative analysis of plant and animal calcium signal transduction element using plant full-length cDNA data. Mol Biol Evol 2004, 21:1855-1870. 39. Collins SR, Meyer T: Evolutionary origins of STIM1 and STIM2 within ancient Ca2+ signaling systems. Trends Cell Biol 2011, 21:202-211. 40. Hetherington AM, Brownlee C: The generation of Ca2+ signals in plants. Annu Rev Plant Biol 2004, 55:401-427. 41. Ma¨ser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D et al.: Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol 2001, 126:1646-1667. 42. Talke IN, Blaudez D, Maathuis FJ, Sanders D: CNGCs: prime targets of plant cyclic nucleotide signalling? Trends Plant Sci 2003, 8:286-293. 43. Ward JM, Ma¨ser P, Schroeder JI: Plant ion channels: gene families, physiology, and functional genomics analyses. Annu Rev Physiol 2009, 71:59-82. 44. Frietsch S, Wang YF, Sladek C, Poulsen LR, Romanowsky SM, Schroeder JI, Harper JF: A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen. Proc Natl Acad Sci U S A 2007, 104:14531-14536. 45. Chaiwongsar S, Strohm AK, Roe JR, Godiwalla RY, Chan CW: A cyclic nucleotide-gated channel is necessary for optimum fertility in high-calcium environments. New Phytol 2009, 183:76-87. 46. Ma W, Berkowitz GA: Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades. New Phytol 2011, 190:566-572. 47. Tunc-Ozdemir M, Tang C, Ishka MR, Brown E, Groves NR, Myers CT, Rato C, Poulsen LR, McDowell S, Miller G et al.: A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development. Plant Physiol 2013, 161:1010-1020. 48. Tunc-Ozdemir M, Rato C, Brown E, Rogers S, Mooneyham A, Frietsch S, Myers CT, Poulsen LR, Malho R, Harper JF: Cyclic nucleotide gated channels 7 and 8 are essential for male reproductive fertility. PLOS ONE 2013, 8:e55277. 49. Gao QF, Fei CF, Dong JY, Gu LL, Wang YF: Arabidopsis CNGC18 is a Ca2+-permeable channel. Mol Plant 2014, 7:739-743. 50. Zhou L, Lan W, Jiang Y, Fang W, Luan S: A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth. Mol Plant 2014, 7:369-376. 51. Kim SA, Kwak JM, Jae SK, Wang MH, Nam HG: Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant Cell Physiol 2001, 42:74-84. 52. Kang J, Turano FJ: The putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of carbon and nitrogen metabolism in Arabidopsis thaliana. Proc Natl Acad Sci U S A 2003, 100:6872-6877. 53. Kang J, Mehta S, Turano FJ: The putative glutamate receptor 1.1 (AtGLR1.1) in Arabidopsis thaliana regulates abscisic acid biosynthesis and signaling to control development and water loss. Plant Cell Physiol 2004, 45:1380-1389. 54. Qi Z, Stephens NR, Spalding EP: Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiol 2006, 142:963-971. 55. Stephens NR, Qi Z, Spalding EP: Glutamate receptor subtypes evidenced by differences in desensitization and dependence on the GLR3.3 and GLR3.4 genes. Plant Physiol 2008, 146:529-538. 56. Cho D, Kim SA, Murata Y, Lee S, Jae SK, Nam HG, Kwak JM: Deregulated expression of the plant glutamate receptor homolog www.sciencedirect.com

Signaling in cells and organisms Steinhorst and Kudla 21

AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure. Plant J 2009, 58:437-449. 57. Price MB, Jelesko J, Okumoto S: Glutamate receptor homologs in plants: functions and evolutionary origins. Front Plant Sci 2012, 3:235. 58. Vincill ED, Clarin AE, Molenda JN, Spalding EP: Interacting glutamate receptor-like proteins in phloem regulate lateral root initiation in Arabidopsis. Plant Cell 2013, 25:1304-1313. 59. Michard E, Lima PT, Borges F, Silva AC, Portes MT, Carvalho JE, Gilliham M, Liu LH, Obermeyer G, Feijo´ JA: Glutamate receptorlike genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine. Science 2011, 332:434-437. 60. Vincill ED, Bieck AM, Spalding EP: Ca2+ conduction by an amino acid-gated ion channel related to glutamate receptors. Plant Physiol 2012, 159:40-46. 61. Manzoor H, Kelloniemi J, Chiltz A, Wendehenne D, Pugin A, Poinssot B, Garcia-Brugger A: Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis. Plant J 2013, 76:466-480. 62. Li F, Wang J, Ma C, Zhao Y, Wang Y, Hasi A, Qi Z: Glutamate receptor-like channel3.3 is involved in mediating glutathionetriggered cytosolic calcium transients, transcriptional changes, and innate immunity responses in Arabidopsis. Plant Physiol 2013, 162:1497-1509. 63. Choi WG, Toyota M, Kim SH, Hilleary R, Gilroy S: Salt stress induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants. Proc Natl Acad Sci U S A 2014, 111:6497-6502. This work is of outstanding importance because it provides a detailed report about the occurrence, propagation and consequences of Ca2+ waves of plants in response to salt stress. Moreover, it also identifies the slow vacuolar channel TPC1 as an important component in this process. 64. Knight MR, Read ND, Campbell AK, Trewavas AJ: Imaging  calcium dynamics in living plants using semi-synthetic recombinant aequorins. J Cell Biol 1993, 121:83-90. This work provides the very first observation of propagating Ca2+ signals in plants. 65. Xiong TC, Ronzier E, Sanchez F, Corratge-Faillie C, Mazars C,  Thibaud JB: Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFPaequorin reporter. Front Plant Sci 2014, 5:43. This work is important because it reports the occurrence of Ca2+ waves in leaves in response to salt stress. 66. Peiter E, Maathuis FJ, Mills LN, Knight H, Pelloux J, Hetherington AM, Sanders D: The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement. Nature 2005, 434:404-408.

71. Bonaventure G, Gfeller A, Proebsting WM, Hortensteiner S, Chetelat A, Martinoia E, Farmer EE: A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis. Plant J 2007, 49:889-898. 72. Bonaventure G, Gfeller A, Rodriguez VM, Armand F, Farmer EE: The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis. Plant Cell Physiol 2007, 48:1775-1789. 73. Beyhl D, Hortensteiner S, Martinoia E, Farmer EE, Fromm J, Marten I, Hedrich R: The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium. Plant J 2009, 58:715-723. 74. Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Van Breusegem F: ROS signaling: the new wave? Trends Plant Sci 2011, 16:300-309. 75. Wrzaczek M, Brosche M, Kangasjarvi J: ROS signaling loops – production, perception, regulation. Curr Opin Plant Biol 2013, 16:575-582. 76. Miller G, Schlauch K, Tam R, Cortes D, Torres MA, Shulaev V,  Dangl JL, Mittler R: The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Sci Signal 2009, 2:ra45. This work is of outstanding importance because it established the concept of ROS waves as a mechanism for long-distance signaling in plants. Moreover, the authors identified the NADPH oxidase RBOHD as a crucial component that is required for the proper generation of a long-distance ROS signal in response to several stimuli 77. Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI: NADPH oxidase AtrbohD and AtrbohF genes function in ROSdependent ABA signaling in Arabidopsis. EMBO J 2003, 22:2623-2633. 78. Torres MA, Jones JD, Dangl JL: Pathogen-induced NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nat Genet 2005, 37:1130-1134. 79. Torres MA, Dangl JL: Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr Opin Plant Biol 2005, 8:397-403. 80. Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE: An expanded palette of genetically encoded Ca2+ indicators. Science 2011, 333:1888-1891.

67. Ward JM, Schroeder JI: Calcium-activated K+ channels and calcium-induced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. Plant Cell 1994, 6:669-683.

81. Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S: Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 2006, 3:281-286.

68. Pottosin II, Tikhonova LI, Hedrich R, Schonknecht G: Slowly activating vacuolar channels can not mediate Ca2+-induced Ca2+ release. Plant J 1997, 12:1387-1398.

82. Matzke AJ, Matzke M: Membrane ‘‘potential-omics’’: toward voltage imaging at the cell population level in roots of living plants. Front Plant Sci 2013, 4:311.

69. Bewell MA, Maathuis FJ, Allen GJ, Sanders D: Calcium-induced calcium release mediated by a voltage-activated cation channel in vacuolar vesicles from red beet. FEBS Lett 1999, 458:41-44.

83. Farmer EE, Gasperini D, Acosta IF: The squeeze cell hypothesis  for the activation of jasmonate synthesis in response to wounding. New Phytol 2014 http://dx.doi.org/10.1111/nph.12897. This paper is very interesting. The authors propose a model for responses in which rapid axial changes in xylem hydrostatic pressure in response to wounding would quickly travel through the vasculature and induce slower radially dispersed pressure changes that activate GLR receptors for Ca2+ mobilization.

70. Stephan AB, Schroeder JI: Plant salt stress status is transmitted systemically via propagating calcium waves. Proc Natl Acad Sci U S A 2014, 111:6126-6127.

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Current Opinion in Plant Biology 2014, 22:14–21