Calcium signaling during reproduction and biotrophic fungal interactions in plants.

Molecular Plant Review Article

Calcium Signaling during Reproduction and Biotrophic Fungal Interactions in Plants Junyi Chen1, Caroline Gutjahr2, Andrea Bleckmann1 and Thomas Dresselhaus1,* 1

Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universita¨tsstraße 31, D-93053 Regensburg, Germany

2

Faculty of Biology Genetics, Biocenter Martinsried, University of Munich (LMU), Grosshaderner Strasse 2-4, D-82152 Martinsried, Germany

*Correspondence: Thomas Dresselhaus ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.01.023

ABSTRACT Many recent studies have indicated that cellular communications during plant reproduction, fungal invasion, and defense involve identical or similar molecular players and mechanisms. Indeed, pollen tube invasion and sperm release shares many common features with infection of plant tissue by fungi and oomycetes, as a tip-growing intruder needs to communicate with the receptive cells to gain access into a cell and tissue. Depending on the compatibility between cells, interactions may result in defense, invasion, growth support, or cell death. Plant cells stimulated by both pollen tubes and fungal hyphae secrete, for example, small cysteine-rich proteins and receptor-like kinases are activated leading to intracellular signaling events such as the production of reactive oxygen species (ROS) and the generation of calcium (Ca2+) transients. The ubiquitous and versatile second messenger Ca2+ thereafter plays a central and crucial role in modulating numerous downstream signaling processes. In stimulated cells, it elicits both fast and slow cellular responses depending on the shape, frequency, amplitude, and duration of the Ca2+ transients. The various Ca2+ signatures are transduced into cellular information via a battery of Ca2+-binding proteins. In this review, we focus on Ca2+ signaling and discuss its occurrence during plant reproduction and interactions of plant cells with biotrophic filamentous microbes. The participation of Ca2+ in ROS signaling pathways is also discussed. Key words: pollen tube, fertilization, calcium, fungal hyphae, mycorrhiza, compatibility Chen J., Gutjahr C., Bleckmann A., and Dresselhaus T. (2015). Calcium Signaling during Reproduction and Biotrophic Fungal Interactions in Plants. Mol. Plant. 8, 595–611.

INTRODUCTION Calcium (Ca2+) signals are core regulators in many crucial and sophisticated cellular processes in eukaryotic organisms. In plants, transient increases in cytosolic free Ca2+ ([Ca2+]cyt) are stimulated during various environmental and developmental processes including root growth, stomatal aperture, circadian rhythm, abiotic stress responses, plant–pathogen interactions, symbiosis, and fertilization mechanisms. The [Ca2+]cyt increases in plant cells often appear as transient spiking or repetitive oscillations where the amplitude, frequency, shape, and duration of the Ca2+ transients are determined by the specific stimulus. Thereafter, the stimulus-specific Ca2+ patterns, referred to as Ca2+ signatures, are transduced into cellular information to regulate proper responses (Sanders et al., 2002; McAinsh and Pittman, 2009). Ca2+ signatures are perceived and translated by a battery of signal receivers, Ca2+-sensor proteins that contain Ca2+-binding EF-hand motifs. Arabidopsis thaliana, for example, possesses at least 250 EF-hand proteins. One hundred of these have been classified as Ca2+ sensor proteins (Day et al., 2002; Hashimoto and Kudla, 2011). The complex array of Ca2+

sensors thus provide a reliable molecular basis for robust and flexible signal processing and therefore contribute to the stimulus specificity of Ca2+ signaling. Plant cells keep [Ca2+]cyt levels very low at about 100–200 nM, otherwise Ca2+ would form insoluble precipitates with phosphate both in the cytoplasm and nucleoplasm. Ca2+ is constantly pumped out of the cell or into intracellular compartments, which allows the establishment of membrane Ca2+ gradients in the magnitude of 103 to 105 times due to a Ca2+ concentration of 10–104 mM inside cellular organelles or in the extracellular environment (Berridge et al., 2000; Konrad et al., 2011). The plant cell wall represents the major storage compartment for Ca2+ with enormous binding capacities for this cation, especially by de-esterified pectin chains. Upon secretion to the cell wall, pectin molecules are de-esterified by pectin methylesterase (PME) and become cross-linked to each other via Ca2+. Pectin cross-linked

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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Calcium Signaling in Reproductive and Biotrophic Interactions

by Ca2+ behaves as a rigidity gel, and the interplay between PME and its inhibitor PMEI regulates cell wall elasticity and the availability of external Ca2+ (Holdaway-Clarke and Hepler, 2003; Bosch and Hepler, 2005). Cells thus actively translocate Ca2+ by membrane transport proteins into specific cellular compartments, especially the endoplasmic reticulum (ER), nuclear envelope, and vacuole(s), to maintain the membrane Ca2+ gradient and to shape Ca2+ transients. Importantly, the extremely low [Ca2+]cyt, the high membrane Ca2+ gradient, and the effective Ca2+ transport system represent prerequisites for an effective signaling machinery. As a consequence, very rapid elevation of [Ca2+]cyt by Ca2+ influx through activated cell surface- or organelle-located Ca2+-permeable channels is capable of quickly triggering specific physiological responses. Moreover, even very small changes in [Ca2+]cyt are sufficient to activate Ca2+-binding proteins such as calmodulin (CaM) and Ca2+-dependent protein kinases (CDPK). These modulate the activity of a large number of downstream effectors, which finally regulate cellular processes leading, for example, to cell division, expansion, differentiation, or even to cell death, in response to various stimuli such as pathogen attack, symbiosis, or pollen tube growth (Harper and Harmon, 2005; Dumas and Gaude, 2006; Lecourieux et al., 2006). Ca2+ signaling in plants functions in local and systemic communication. Rapid systemic signaling is activated in response to different stimuli including mechanical force, pathogen infection, and abiotic stresses and results in systemic propagation of Ca2+ and reactive oxygen species (ROS) waves. This allows plant cells to transmit long-distance signals via cell-to-cell communication (Steinhorst and Kudla, 2013b; Gilroy et al., 2014). In this review, we focus on local, short-distance signaling during direct contacts between cells, especially on recent research regarding calcium signaling in reproductive and fungal interactions. Based on this, we highlight similarities and differences among these two systems to further our understanding of the cellular processes in the Ca2+ signaling network and the strong overlap of reproductive and defensive signaling mechanisms.

THE MOLECULAR PLAYERS OF Ca2+ SIGNALING Ca2+ signaling occurs after Ca2+ permeable channels located in the membrane are activated to mediate Ca2+ influx into the cytoplasm. Ca2+ efflux mediated by pumps and exchangers then acts to reduce the magnitude and duration of Ca2+ elevation and restores the cells to the resting state. The concerted action of calcium channels, pumps, and exchangers coordinately regulate the formation of stimulus-specific Ca2+ signatures, which are sensed and transduced by a toolkit of Ca2+ binding proteins that decode the Ca2+ signature into defined phosphorylation or transcriptional changes thereby leading to downstream physiological responses. Thus, the Ca2+ transporters and the complex set of Ca2+ binding proteins constitute the major molecular components of Ca2+ signaling pathways in plants (see Figure 1 for an overview).

Ca2+-Permeable Channels: Initiating Cytosolic Free Ca2+ Elevation As introduced above, the transient elevation of [Ca2+]cyt is achieved via Ca2+ fluxes from extracellular and intracellular 596


stores into the cytosol through Ca2+-permeable channels in the plasma membrane and endomembrane system, respectively. According to their activation mechanisms, these Ca2+-permeable channels can be classified as hyperpolarization-activated calcium channels (HACCs), depolarization-activated calcium channels (DACCs), and voltage-independent calcium channels (VICCs). Typically, channels of cells such as pollen tubes with a resting potential of approximately 80 to 100 mV (e.g. Amien et al., 2010) possess a threshold for opening around this voltage, which means that these channels are poised to open. DACCs are activated at a less negative membrane voltage, while HACCs dominantly mediate Ca2+ influx/release at a higher negative membrane potential. It has been clearly demonstrated that DACCs, HACCs, and VICCs can coexist in the plasma membrane of higher plant cells (Swarbreck et al., 2013) indicating that plant cells are competent for Ca2+ influx across a wide range of membrane potentials. In addition to membrane voltage, these channels are usually also activated by other factors, such as cAMP/cGMP, [Ca2+]cyt, phosphorylation, and ROS (Dodd et al., 2010; Swarbreck et al., 2013). Similar to bow-tie network architecture in neural network computing (Csete and Doyle, 2004), it is obvious that Ca2+ transporters connect diverse inputs (stimuli) and outputs (Ca2+ signatures) using defined parameters and thus represent one class of core intermediates in Ca2+ signaling. The molecular identity of Ca2+-specific influx channels remained unknown or controversial for a long time (Steinhorst and Kudla, 2013a). Like vacuolar membrane-localized two-pore channels (TPCs) forming Ca2+- and voltage-dependent cation channels (Furuichi et al., 2001; Peiter et al., 2005), most channels that transport are also permeable to other monovalent or divalent cations, thus do not bind Ca2+ specifically. Various classes of Ca2+-permeable channels are described below. Cyclic Nucleotide-Gated Channels In Arabidopsis, 20 genes have been identified to encode cyclic nucleotide-gated channels (CNGCs), which can be modulated by cyclic nucleotides (cAMP and/or cGMP) (Ma¨ser et al., 2001; Kaplan et al., 2007). Besides their Ca2+-permeable property, some CNGCs are also permeable to monovalent or other divalent cations. Plant CNGCs are thought to consist of four subunits forming a non-selective pore in the center (Kaplan et al., 2007). Each subunit contains six transmembrane domains (TMDs) with both N and C termini directed toward the cytoplasm. A cyclic nucleotide binding (CNB) domain overlaps with a CaM binding domain in the large cytosolic C-terminal region. This molecular structure introduces a feedback regulation between Ca2+/CaM- and cyclic nucleotide-regulated CNGC activity as binding affinity of cyclic nucleotide to the CNB domain would be reduced in the presence of Ca2+/CaM and vice versa. Most CNGCs studied so far localize to the plasma membrane (Jammes et al., 2011; Wang et al., 2013), indicating that these channels mainly mediate Ca2+ influx from extracellular stores (cell walls in plants). Glutamate Receptor-like Channels The glutamate receptor (GLR) family of transporters also consists of 20 gene members in Arabidopsis, each of which encodes a protein with three transmembrane domains. Similar to CNGCs, GLRs form non-selective ligand-gated channels, which are permeable to Ca2+, but additionally to the monovalent cations


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Figure 1. Generalized Model of Ca2+ Signaling Occurring in Cells Involved in Plant Reproduction and Fungal Invasion. (A) Model showing the molecular players involved in Ca2+signaling of a reproductive plant cell (e.g. pollen tube, papilla cell, synergid cell, female gametes). Only protein classes are indicated. See the text for individual members of protein classes that have been functionally characterized. [Ca2+]cyt spikes are induced by external ligands perceived by cell surface receptors (shown on the left), which induce opening of [Ca2+]cyt channels, partly via ROS production, resulting in Ca2+spikes after influx from external stores (e.g. cell wall and burst cells). The involvement of Ca2+ release from internal stores (shown on the right: vacuole, ER, or nuclear envelope) is unclear. A battery of Ca2+ binding proteins (e.g. CaM/CML, CBLs, CDPKs, ABPs) decode the spiking pattern and activate fast responses (e.g. ROS production, vesicle secretion, actin depolymerization, Ca2+ efflux, and inhibition of Ca2+ influx) or induce slow responses in the nucleus via modulation of the gene expression pattern. (B) Model showing the molecular players in Ca2+ signaling discussed in the main text during plant cell invasion by pathogens (top) and symbionts (bottom). See text for a detailed description and references. Many signaling events shown in (A) likely occur also during infection of plant cells by symbiotic fungi and during infection by pathogenic filamentous microbes (fungi and oomycetes; see also Seybold et al., 2014). Top: during defense, microbe associated molecular patterns (MAMPS) are perceived by receptor-like kinases, their co-receptors, and extracellular receptor proteins, which leads to the activation of Ca2+channels and a transient increase in [Ca2+]cyt. The increase in [Ca2+]cyt activates CDPKs, which activate NADPH oxidases by phosphorylation. The resulting ROS burst can also trigger Ca2+channels, which again leads to [Ca2+]cyt elevations. Bottom: the molecular players required for generation and interpretation of nuclear Ca2+ spiking during cell infection by symbiotic arbuscular mycorrhiza (AM) fungi. Fungus-released chitin oligomers are perceived by receptor-like kinases that probably trigger the generation of a yet unknown secondary messenger that induces nuclear Ca2+ spiking. This requires currently unknown Ca2+ channels and potassium-conductive counter ion channels. Ca2+ spiking is interpreted by nuclear-localized Ca2+ and CaM-dependent kinases (CCamK), which phosphorylate transcription factors.

Na+ and K+. Unlike CNGCs, the two putative ligand-binding motifs in GLRs are predicted to face the extracellular space. Glutamate as well as the five amino acids, glycine, alanine, serine, aspartic acid, and cysteine, and even glutathione, have been reported to activate GLR3.3 with different efficiencies (Qi et al., 2006). Recently, D-serine likely generated in ovules was reported to regulate GLR1.2 activity in pollen tubes and thereby modulate their growth behavior (see below; Michard et al., 2011). GLR activation by exogenous amino acids (e.g. glutamate) triggers [Ca2+]cyt elevation resulting in plasma membrane depolarization (Dennison and Spalding, 2000; Meyerhoff et al., 2005). The Ca2+ transient in the cytoplasm thereafter activates Ca2+-regulated kinases, which in turn may regulate GLR activities through 14-3-3 motifs in GLRs (Chang et al., 2009; Latz et al., 2013). Therefore, activation or deactivation of GLRs by Ca2+-regulated kinases seem to represent a mechanism of positive or negative feedback regulation, respectively.

Mechanosensitive Channels: MSLs, SACs, and OSCAs Ca2+ signaling also appears to be key for plants to sense mechanical stimuli and osmolarity during increased membrane tension. Cytosolic Ca2+ elevation was observed after mechanical stimulation and mechano-sensitive Ca2+-permeable channels were suspected to mediate Ca2+ influx in this process (Knight et al., 1991). In Arabidopsis, 10 members of the MscS-like (MSL) family were identified based on their similarity to bacterial mechanosensitive channels. However, the Ca2+-permeable properties of plant MSLs have not yet been determined (Kurusu et al., 2013). Moreover, various msl mutants do not show obvious defects in mechanosensitivity, suggesting the existence of other mechanosensing members in this gene family or a major role of stretch-activated Ca2+ channels (SACs) in mechanosensitivity. Two stretch-activated Ca2+-permeable channels named MCA1 (mid1-complementing activity 1) and MCA2 were characterized Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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in more detail in Arabidopsis. Both channels are capable of complementing the yeast mid1 mutant encoding a stretch-activated Ca2+ channel (Nakagawa et al., 2007; Yamanaka et al., 2010). MCAs are predicted to contain two transmembrane domains with both N and C termini facing the cytosol. The Ca2+ binding EF-hand domain in the N-terminal region is important for Ca2+uptake (Nakano et al., 2011). Like GLRs, all MCAs characterized so far are localized in the plasma membrane and appear to mediate Ca2+ influx from extracellular stores. A novel class of mechano-stimulated Ca2+permeable channels termed reduced hyperosmolality-induced [Ca2+]I increase (OSCA) was just recently described in Arabidopsis (Yuan et al., 2014; Hou et al., 2014). OSCA1.1 and 1.2 are capable of conducting Ca2+ and were shown to be involved in environmental osmo-sensing, especially in stomata and roots. However, like GLRs and CNGCs, OSCAs form a large gene family conserved throughout the eukaryotic kingdom (Edel and Kudla, 2015) and its members may also function in many other processes including reproduction and defense. ROS Responsive Ca2+-Permeable Channels ROS are the byproducts of aerobic metabolism and they can cause damage to cells. Recent reports have indicated that similar to Ca2+, plants have evolved ROS as major signaling molecules regulating various processes including seed germination, growth, biotic and abiotic stress responses, and programmed cell death (PCD) (Bailey-Serres and Mittler, 2006; Gechev et al., 2006). Interestingly, ROS are also involved in Ca2+ signaling pathways as some Ca2+ channels are identified to be ROS responsive. The Shaker-like STELAR K+ OUTWARD RECTIFIER (SKOR), for example, is an H2O2 responsive K+ channel with Ca2+ permeability that can mediate Ca2+ influx at the plasma membrane (Gaymard et al., 1998; Garcia-Mata et al., 2010). The wellstudied annexins can function as Ca2+-permeable channels in vivo as well as in planar lipid bilayer (PLB), and their Ca2+ conductance capacity can be activated by hyperpolarization and extracellular ROS molecules such as hydroxyl radicals (HO$) (Laohavisit et al., 2010, 2012).

Calcium Pumps and Antiporters: Terminating [Ca2+]cyt Elevation and Shaping Ca2+ Signatures Cells need to efficiently translocate Ca2+ out of the cytosol not only to maintain cell viability but also to shape distinct Ca2+ signatures after Ca2+-releasing events. Extrusion of Ca2+ from the cytoplasm is achieved by two energized systems: calcium pumps and calcium-proton antiporters (also known as cation exchangers, CAXs). There are two types of classical calcium pumps: ACAs (autoinhibited or PIIB-type Ca2+ ATPases) and ECAs (ER-type or PIIA-type Ca2+ ATPases). It was suggested that ACAs represent the key players in Ca2+ signaling while ECAs are involved in Ca2+ delivery for Ca2+ requiring activities in various intracellular compartments (Dodd et al., 2010). ACAs contain an N-terminal cytosolic domain capable of binding Ca2+/CaM. This interaction activates Ca2+ pumping and thus results in [Ca2+]cyt reduction. One impressive study conducted in Physcomitrella patens showed that in comparison with wildtype plants, which exhibited a transient Ca2+ signature after salt stress, aca mutant plants showed a sustained [Ca2+]cyt elevation, reduced up-regulation of stress responsive genes, and less sodium stress tolerance (Qudeimat et al., 2008). Many other 598


studies with aca mutants as well as cax mutants demonstrated that fine-tuned Ca2+ extrusion is as important as Ca2+ influx and that the Ca2+ exporters (including CAXs, ECAs, and ACAs) are required for Ca2+ homeostasis and correct Ca2+ signaling (Bose et al., 2011).

Ca2+-Sensor Proteins: Sensing and Decoding Ca2+ Signals The stimulus-specific [Ca2+]cyt elevations or signatures are perceived and decoded by calcium sensor proteins that transduce various Ca2+ signals into defined downstream cellular responses. All Ca2+-sensor proteins contain at least one helixloop-helix domain, namely EF-hand motif, which mediates Ca2+ and sensor protein binding. Depending on the nature of the response domain (either a kinase or transcription regulation domain), the Ca2+ sensor proteins are classified into sensor relay proteins or sensor responders. Sensor Relay Proteins: Connecting Ca2+ Signals with Responders Sensor relay proteins possess Ca2+ binding motifs but lack other effector domains. They can effectively bind Ca2+ and transduce Ca2+ signals through induced conformational changes that facilitate their interaction with target effectors. The most ubiquitous Ca2+ sensor relay proteins found in all eukaryotes are CaMs as well as CaM-like (CML) proteins. CaMs and CMLs convert Ca2+ signatures, for example, into transcriptional responses by modulating CaM binding transcription factors (CAMTAs) (Finkler et al., 2007; Doherty et al., 2009). In addition to CaMs and CMLs, calcineurin B-like proteins (CBLs) are also considered as sensor relay proteins as they represent Ca2+ sensors without any enzymatic function. CBLs can bind Ca2+ with their four conserved EF hands. In order to transmit the Ca2+ signals, CBLs additionally need to interact with members of the CIPKs (CBL-interacting protein kinases) family to generate enzymatically active complexes (Shi et al., 1999). The 10 CBLs present in Arabidopsis are able to interact with all 26 CIPKs. Remarkably, CIPKs are capable of interacting with different CBLs at a defined location within the same cell (e.g. plasma membrane or tonoplast), enabling the generation of a flexible and complex network for spatially specific and efficient Ca2+ et al., 2010). responses (Batistic Sensor Responders: Translating Ca2+ Signals Directly into Responses In contrast to sensor relay proteins, sensor responders like Ca2+dependent protein kinases (CDPKs or CPKs) combine a sensing domain (for Ca2+ binding and Ca2+-induced conformational changes) with a response domain (e.g. a kinase domain) within a single protein thus representing versatile Ca2+ sensors that can directly transduce Ca2+ signals via phosphorylation of target proteins (Cheng et al., 2002). In summary, Ca2+ sensor proteins translate diverse inputs (Ca2+ signatures) into outputs (e.g. protein phosphorylation or transcriptional regulation) using defined parameters. Similar to Ca2+ transporters, they represent a second layer of core intermediates in the architecture and hierarchy of Ca2+ signaling networks. Plants possess a large repertoire of Ca2+ sensors. In Arabidopsis, for example, there exist 7 CaMs, 50 CMLs, 10 CBLs, 26 CIPKs, and 34 CDPKs (Steinhorst and Kudla, 2013a). The high number of Ca2+ sensor complexes, their different Ca2+ binding affinity,

Calcium Signaling in Reproductive and Biotrophic Interactions and cell-specific and subcellular localization patterns thus provide a robust and sophisticated system for efficient and precise decoding of information presented in the diverse Ca2+ signatures.

Cross-Talk between Signaling Pathways Ca2+ signaling does not function as a separate network. In fact, many processes in other signaling pathways are Ca2+ sensitive and various small molecules are capable of regulating or modulating Ca2+ signaling pathways. These reciprocal interactions facilitate information flow to and from other signaling pathways, which in turn reinforce the universality and versatility of Ca2+ signaling. Ca2+ signaling is, for example, integrated in phospholipase C (PLC)-dependent signaling pathways. PLCs have long been known to play a key regulatory role in phosphoinositide signal transduction pathways. Plant PLCs belong to the so-called PLCz class, which contain an EF-hand domain and thereby are regulated by Ca2+ (Munnik and Testerink, 2009). In addition, phosphatidic acid (PA) has emerged as an important signaling lipid in plant. PA can be rapidly and transiently produced after various biotic and abiotic stresses involving the enzyme phospholipase D (PLD). The majority of plant PLDs contain a C2 (Ca2+ and lipid binding) domain. The C2 domain is thought to be involved in Ca2+-dependent phospholipid binding and thus regulates membrane targeting and the activity of PLD in a Ca2+-dependent manner (Kolesnikov et al., 2012). Moreover, Ca2+ participates in ROS signaling pathways (Figure 1). Plant ROS are produced by respiratory burst oxidase homolog (RBOH) NADPH oxidases, which possess two Ca2+-binding EF hands. Binding of Ca2+ activates RBOH and as a consequence enhances cellular ROS accumulation (Takeda et al., 2008). Studies in potato suggest that two Ca2+ sensors, StCDPK4 and StCDPK5, can phosphorylate NADPH oxidases and thereby positively regulate the production of ROS (Kobayashi et al., 2007). These two Ca2+-dependent RBOH activation mechanisms may work synergistically in regulating ROS production and signaling. ROS on the other hand have been shown to activate several Ca2+-permeable channels, such as SKOR and annexins (Garcia-Mata et al., 2010; Laohavisit et al., 2012). Moreover, in concert with nitric oxide (NO), it appears able to activate guanylyl cyclases to produce cGMP and thereby possibly indirectly activate CNGCs through increases in cGMP levels. The activation of Ca2+-permeable channels then triggers [Ca2+]cyt elevations, which feed back to negatively regulate CNGCs by CaM and at the same time activate Ca2+ pumps such as ACAs, thereby terminating influx and enhancing efflux to reduce [Ca2+]cyt levels (Baekgaard et al., 2005; Kaplan et al., 2007).

CALCIUM IS A KEY REGULATOR OF POLLEN TUBE INVASION AND GROWTH As a ubiquitous and versatile second messenger, Ca2+ plays a key role during the complex reproductive processes occurring in flowering plants (angiosperms). Ca2+ signaling (Figure 1A) occurs during pollen germination and interaction with the

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papillae cells of the stigma, during pollen tube growth through the sporophytic maternal tissues of the female flower organs, and during its arrival at the ovule leading to sperm cell release (see below). Nearly 50 years ago, Brewbaker and Kwack (1963) demonstrated that Ca2+ was essential for pollen germination and pollen tube growth. Since then, the critical role of Ca2+for polar cell elongation has been extensively studied in this excellent tip-growing model system.

Oscillatory Tip-Focused Ca2+ Gradient in a Growing Pollen Tube Jaffe et al. (1975) were the first to observe a tip-focused Ca2+ gradient (Figure 2A) and an extracellular Ca2+ influx at the tip of lily (Lilium longiflorum) pollen tubes. Nowadays, it is well established that all growing pollen tubes possess a characteristic steep Ca2+ gradient at their extreme apex, in which the Ca2+ concentration extends from 2 to 10 mM in close proximity to the plasma membrane to a basal level of 20– 200 nM within 20 mm from the tube tip. Tip-directed Ca2+ influx from the extracellular space represents the major source of this Ca2+ gradient and the entry of Ca2+ ions in the pollen tube apex frequently exhibits an oscillatory pattern (Pierson et al., 1994; Pierson et al., 1996). It was further reported that the [Ca2+]cyt gradient oscillates with the same period of about 15–60 s in phase with pollen tube growth, while tip-localized Ca2+ influx lags by about 11 s in pollen tube model systems such as lily (Holdaway-Clarke et al., 1997). However, the occurrence of Ca2+ oscillations in pollen tubes are controversially discussed (Steinhorst and Kudla, 2013a) and might appear mainly in pollen tubes of species showing pronounced periodic growth such as lily. Oscillation frequency in pollen tubes of Arabidopsis or tobacco grown in vitro was very fast (about five spikes per minute) with low amplitude, but hardly detectable in in vivo growing tubes (Iwano et al., 2004; Iwano et al., 2009; see also Figure 2E). Nevertheless, this indicated that SAC channels are likely involved in Ca2+ influx and the maintenance of the Ca2+ gradient. SAC activities were found to be present at the extreme apex of the tube and at the aperture of the pollen grain. During pollen tube elongation, the apical plasma membrane would deform due to the force of turgor pressure, and this would activate SACs to mediate Ca2+ influx (Dutta and Robinson, 2004; Hepler and Winship, 2010). Several studies have revealed CNGCs and GLRs to be responsible for Ca2+ influx in pollen tubes (Frietsch et al., 2007; Michard et al., 2011). To shape the tip-focused Ca2+ gradient and to maintain low [Ca2+]cyt in the pollen tube shank, it is also important that Ca2+ ions are either efficiently expelled at the subapical region toward the extracellular space or sequestered into intracellular compartments (e.g. the vacuole, ER, or nuclear envelope), which are lacking in the so-called clear zone of the tube apex showing high [Ca2+]cyt (see also below). Several Ca2+ pumps and antiporters located in the endomembrane, such as ACA2, ECA1, and CAXs, are expressed in pollen tubes (M. Woriedh and T. Dresselhaus, unpublished results) and likely function in Ca2+ sequestration. The CaM-dependent Ca2+ pump ACA9 uniformly distributed at the pollen tube plasma membrane is assumed to expel Ca2+ to the apoplast and was shown to be required for normal pollen tube growth and fertilization (Schiøtt et al., 2004). Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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Figure 2. Comparison of [Ca2+]cyt Signatures in Gametophytic Cells during the Process of Double Fertilization and in Sporophytic Cells during Fungal Infection. (A–D) False color ratio metric images of relative [Ca2+]cyt in reproductive cells expressing the calcium sensor CerTN-L15 using cell type-specific promoters (MYB98, pollen; EC1.1, egg cell; DD65, central cell; and ARO1, pollen). Scale bars, 20 mm. (A) Pollen grains and a Growing pollen tube, (B) synergid cells, (C) egg cell, and (D) central cell of Arabidopsis thaliana. For marker line details, please see Denninger et al., 2014. (E) [Ca2+]cyt signatures in a growing pollen tube, synergid cells, egg cell, and central cell during the process of double fertilization. Displayed are normalized ratio metric changes in CerTN-L15 in respect of the event of pollen tube burst (0 min). Arrow, first physical contact of pollen tube and synergid cells; star, pollen tube burst. In Arabidopsis the growing pollen tube does not display significant [Ca2+]cyt oscillation. In synergids cells, [Ca2+]cyt starts oscillation after physical pollen tube contact. Shortly before pollen tube burst, the high [Ca2+]cyt level is maintained in the cell followed by a [Ca2+]cyt spike, which overlaps with pollen tube burst (Supplemental Movie 1). In the egg cell, two distinct [Ca2+]cyt elevations can be monitored. The first narrow and sharp spike coincides with pollen tube burst/sperm cell arrival (Supplemental Movie 2), whereas the second peak is broader and is correlated to gamete fusion. Please note that deformation of egg cells by the growing pollen tube leads to a focal plane shift associated with a signal increase (arrowhead) but no spiking. The central cell shows only one [Ca2+]cyt elevation in response to pollen tube burst (Supplemental Movie 3). (F and G) Redrawn measurements of [Ca2+]nucl spiking occurring in root tissue during arbuscular mycorrhizal fungal infection of M. truncatula (after Sieberer et al., 2012). Penetration of epidermis cells is associated with a high frequency of Ca2+cyto spikes (cell e1). Low-frequency [Ca2+]nucl spiking is observed in the underlying cortical cell c1, in which the nucleus has migrated to the future site of hyphal entry. Neighboring cells (c2) do not show any increase in [Ca2+]nucl level.

Besides the tip-focused Ca2+ gradient, other ionic gradients such as K+, Cl and H+ also exist at the tip of growing pollen tubes. The H+ gradient is characterized by an alkaline band at the base of the clear zone and an acidic region at the extreme apex, and it may be closely related to polarized pollen tube growth (Feijo´ et al., 1999). The importance of a K+ gradient was revealed by the Shaker K+ channel loss-of-function mutant spik, which shows 600


disrupted K+ uptake and impaired pollen tube growth (Mouline et al., 2002). Notably, the anion Cl exhibits reversed flux direction compared with the cations Ca2+, H+, and K+, as it flows out at the apex and enters along the tube. Cl efflux at the pollen tube tip has a role in pollen tube growth and cell volume regulation (Zonia et al., 2002). Like Ca2+, the oscillatory influx of H+ and K+, and efflux of Cl at the tip region are in

Calcium Signaling in Reproductive and Biotrophic Interactions phase with oscillatory growth of the pollen tube and appear to work synergistically in a network to regulate pollen tube ion homeostasis and growth (Holdaway-Clarke and Hepler, 2003).

Tip-Focused Ca2+ Gradient Controls Polar Tip Growth In pollen grains, a tip-focused Ca2+ gradient is established at the potential germination site soon after pollen hydration (Iwano et al., 2004). Inhibition by application of Ca2+ channel blockers (e.g. nifedipine and La3+) abrogates the Ca2+ gradient and prevents either pollen germination or pollen tube elongation (Obermeyer and Weisenseel, 1991; Rathore et al., 1991; Iwano et al., 2004). Interestingly, localized apical increase of cytosolic free Ca2+ leads to pollen tube reorientation toward the site of higher [Ca2+]cyt (Rathore et al., 1991), suggesting that the tipfocused Ca2+ gradient is not only necessary for pollen germination and pollen tube elongation but is also important for directing pollen tube growth orientation. Based on these intriguing studies, it is tempting to understand how Ca2+ ions actually work to regulate pollen tube growth. It is well established that the apical 15–20 mm region of the growing pollen tube is characterized by the cone-shaped apical clear zone containing abundant secretory vesicles (Rathore et al., 1991). Large organelles are specifically excluded from this region, providing a relatively clear volume for vesicle transport and fusion. It is discussed that the elevated [Ca2+]cyt in the tipfocused gradient may attract negatively charged vesicles to promote their transportation, adherence, and fusion with the plasma membrane at the pollen tube tip (Ge et al., 2007). The Ca2+ gradient at the apex thereby facilitates vesicle secretion and contributes to local enhancement of pollen tube growth by directing the site of vesicles secretion. As a consequence, local Ca2+ accumulation defines the orientation of pollen tube growth. In addition to vesicle trafficking, several studies revealed that Ca2+ could control the stabilization or depolymerization of the actin cytoskeleton through modulating actin binding proteins (ABPs). ABPs are evenly distributed in the pollen tube but their activities are highly regulated by [Ca2+]cyt. In general, high [Ca2+]cyt promotes fragmentation (mainly by the action of villin and gelsolin) while low [Ca2+]cyt facilitates polymerization (largely by the activity of profilin) (Kovar et al., 2000; Zhang et al., 2010). The highly concentrated Ca2+ gradient at the pollen tube tip thus induces the fragmentation of F-actin (see also Figure 1A), which provides the basis for subsequent cytoskeleton reorganization and thereby ensures the flexibility of the tube tip to change growth directions. This is important as pollen tubes need to change their growth direction several times to reach the female gametophyte where their cargo, the two sperm cells, is unloaded.

Ca2+ in Pollen-Pistil Interaction and Self-Incompatibility Response Consistent with the necessity of tip-directed Ca2+ influx for pollen germination and tube elongation, abundant Ca2+ was observed at the stigma surface as well as in the apoplastic system of stylar transmitting tissues. Strong increases of [Ca2+]cyt were reported to occur just below the surface of papilla cells of the stigma at the site of pollen grain attachment (Iwano et al., 2004). A first peak occurred soon after pollen hydration and a second elevation after pollen tube protrusion. The strongest increase

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occurred when the tube penetrated into the papilla cell. Recently, the autoinhibited Ca2+-ATPase 13 (ACA13) expressed in the stigmatic papilla cells was shown to function in the export of Ca2+ from papilla cells to compatible pollen grains (intraspecific non-self-pollen) to support germination and invasion (Iwano et al., 2014). Ca2+ also plays a key role in preventing self-fertilization (or inbreeding). More than half of all flowering plant species investigated show self-incompatibility (SI) responses (Takayama and Isogai, 2005). Pistils are sterile to their own pollen but fertile with pollen from other individuals of the same species. In poppy (Papaver rhoeas), for example, SI response occurs within minutes after self-pollen deposits on the stigma. A series of studies suggest a model of a two-step SI inhibition process. Within seconds after contact with an incompatible pollen tube, the mobile stigmatic S-protein named PrsS (a CRP) activates the male S-receptor PrpS (a small novel transmembrane protein) at the plasma membrane of the pollen tube and triggers Ca2+ influx along the shank of the pollen tubes resulting in [Ca2+]cyt increases from 210 nM to 1.5 mM for several minutes (Wu et al., 2011). The elevated cytosolic free Ca2+ induces F-actin depolymerization, tip-focused Ca2+ gradient dissipation, and pollen tube growth inhibition within 1–2 min (Franklin-Tong et al., 1997; Geitmann et al., 2000). The first rapid inhibition phase also includes an increase in Ca2+/CaM-dependent phosphorylation of a cytosolic inorganic pyrophosphatase p26, which occurs within 90 s of SI induction. P26 activity is proposed to inhibit pollen tube metabolism (de Graaf et al., 2006). Several minutes after rapid inhibition, a mitogen-activated protein kinase (MAPK), p56, is activated and this may trigger a signaling cascade resulting in inhibition of incompatible pollen tube growth (Rudd et al., 2003). The promotion of caspase (cysteine-aspartic protease)-like activity and subsequent PCD of pollen tubes represent a later irreversible decision-making phase of the SI response (Thomas and FranklinTong, 2004). In conclusion, [Ca2+]cyt increases play a key role during the first rapid inhibition phase and indirectly influence PCD during SI in poppy. In SI pollen tubes, the continuous high levels of [Ca2+]cyt correlate with subsequent PCD. This correlation is also observed in receptive synergid cells during pollen tube reception as described in the following section. Although the role of Ca2+ for pollen tube germination, invasion, and growth is now well established, little is known about decoding and transduction of its signatures. In Arabidopsis, many Ca2+ sensor relay proteins are expressed in pollen, some of which appear specific (Steinhorst and Kudla, 2013a). Of 7 CaMs and 50 CMLs, at least 1 and 18 are strongly and specifically expressed in pollen grains and tubes, respectively. Nine CMLs are pollen specific. From 10 CBLs at least 4 CBLs appear pollen specific. From the complex network of sensor responders, 17 of 34 CDPKs are expressed in pollen grains and tubes of which five are pollen specific and likely also members of the 26 CIPKs.

OSCILLATING CALCIUM WAVES IN SYNERGID CELLS REGULATE POLLEN TUBE BURST AND DEATH Pollen tube reception involves intensive male–female communication to successfully deliver and release the two sperm cells Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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into the female gametophyte for double fertilization (Kessler and Grossniklaus, 2011; Bleckmann et al., 2014). Live cell observation of Ca2+ signatures in the male and female gametophytes in this process has long been prevented as the female gametophyte is deeply embedded in the sporophytic tissue. To circumvent this difficulty, Iwano et al. (2012) employed a genetically encoded Ca2+ sensor, yellow cameleon (YC) 3.60, and a semi-in vivo fertilization system to monitor Ca2+ dynamics in pollen tubes and synergid cells, respectively, allowing them to observe characteristic dynamic changes of Ca2+ levels in these cells during their interaction in vivo. Subsequent studies with modified methods have demonstrated that a Ca2+ dialog mediated by the FERONIA signal transduction pathway controls sperm cell delivery in plants (Ngo et al., 2014). Features of this crucial Ca2+ dialog are revealed and discussed below. When the pollen tube approached the female gametophyte and got into physical contact with the micropylar pole of the synergid cells, the first visible Ca2+ oscillations were initiated in the synergid cells (Figure 2B and 2E; Supplemental Movie 1) with a periodicity of about 100 s at the contact point and spread toward the chalazal pole (Iwano et al., 2012; Denninger et al., 2014; Ngo et al., 2014). While Ngo et al. (2014) reported similar magnitudes and periodicity of Ca2+ elevations in the two synergid cells, Denninger et al. (2014) showed that spiking largely depends on direct interaction between a synergid cell with the pollen tube apex and thus differs between both synergid cells. About 30 min after interaction, [Ca2+]cyt at the pollen tube tip elevated to about four times the level of arrival and the pollen tube started to grow across the receptive synergid cell (RSY) toward the chalazal pole. In the first 1/3 of this phase, the pollen tube growth rate and [Ca2+]cyt remained high, then both decreased and increased again during the last 1/4 of this phase (Ngo et al., 2014). Ca2+ oscillations in the RSY, triggered mainly by the pollen tube apex, flooded the whole cell and were maintained for about 20 min. Finally, [Ca2+]cyt at the pollen tube tip increased up to eight times the level of its arrival and suddenly ruptured. Within 5–10 s, [Ca2+]cyt in the two synergid cells also reached a maximum and returned rapidly to a basal level. Interestingly, the persistent synergid started oscillation again afterwards in the absence of a pollen tube (Ngo et al., 2014). Further investigation revealed that the synergid cell mutants fer, lre, and nta, which showed disrupted pollen tube reception, displayed aberrant Ca2+ patterns in the synergid cells during their interaction with pollen tubes. According to the distinct abnormal Ca2+ signatures in the mutant synergid cells, it was concluded that the receptor-like kinase FER and the GPI-anchored protein LRE are required for initiating Ca2+ oscillations as well as for accurate responses to pollen tube signals, while the CaM binding protein NTA is an important modulator of the magnitude of the Ca2+ signatures in the cells involved in pollen tube reception (Ngo et al., 2014). Moreover, flowers treated with the calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid acetoxymethyl ester (BAPTA-AM) (Kline and Kline, 1992) showed reduction of calcium oscillations in synergid cells and abolishment of pollen tube and synergid cell rupture. A prolonged, frequent, and high level of [Ca2+]cyt oscillation was also reported by Denninger et al. (2014) and correlated with 602


irreversible PCD of synergid cells. Cells showing low and infrequent Ca2+ spiking did not undergo death. Whether frequent and high Ca2+ amplitudes are required to activate, via CDPKs, sufficient NADPH oxidases to produce a ROS burst and thus a hypersensitive response leading to cell death requires further experimentation on the signal decoding and response machinery. In conclusion, the FER-dependent signal transduction pathway seems to be required for correct Ca2+ responses to pollen tube (apex) signals in the synergid cells, and the induced Ca2+ signatures in the RSY, especially the prolonged high level of [Ca2+]cyt, lead to pollen tube burst and PCD of the RSY. The pollen tube signals that activate the FER pathway are not known. However, a small and secreted protein of the RALF (rapid alkalinization factor) subfamily of CRPs was recently shown to interact with the FER receptor in root cells causing phosphorylation of plasma membrane H+-adenosine triphosphatase 2 (AHA2) thus mediating the inhibition of proton transport (Haruta et al., 2014). Pollen tubes express a large number of RALF-encoding genes (M. Woriedh and T. Dresselhaus, unpublished results) suggesting that the ligand(s) of FER exposing synergid cells may include pollen tube secreted RALF proteins. However, this hypothesis needs experimental support. Studies of the FER receptor-like kinase in roots further showed that it interacts with ROP2 GTPases and acts upstream of ROS production (Duan et al., 2010). ROS were indeed also detected in synergid cells, where ROS burst depends on stigma pollination but precedes fertilization (Martin et al., 2013) indicating that ROS production and its dialog with Ca2+ signaling depends on FER. A subsequent study by Duan et al. (2014) confirmed that FER controls the production of high levels of ROS at the entrance to the female gametophyte to induce pollen tube rupture and sperm release for fertilization.

DOES CALCIUM SIGNALING ACTIVATE FEMALE GAMETES? The important role of Ca2+ signaling during fertilization is well characterized in animal model systems. Entry of a sperm cell into the egg cell triggers cytosolic Ca2+ elevation, which acts as a key signal to induce subsequent egg activation events that initiate the development of a new individual (reviewed by Stricker, 1999; Miyazaki and Ito, 2006; Ducibella and Fissore, 2008). In flowering plants, the first evidence of a Ca2+ transient at fertilization was obtained by in vitro fertilization of maize gametes. In a medium containing 5 mM CaCl2, 80% of maize male–female gamete pairs adhered for several minutes and then fused within 10 s in a cell-specific manner (Faure et al., 1994). The gamete adhesion step did not induce any cytosolic Ca2+ variation, whereas the fusion step triggered a [Ca2+]cyt elevation in the fertilized egg cell lasting up to 30 min (Digonnet et al., 1997). Subsequent studies revealed that gamete fusion triggered a Ca2+ influx in close proximity to the sperm entry site with a delay of 1.8 ± 0.6 s. Ca2+ influx subsequently proliferated as a wavefront through the whole egg cell plasma membrane, and then spread homogeneously over the whole egg cell with an average duration of 24.4 min. Moreover, the authors found that Ca2+ influx was a sufficient condition for cell wall formation, which likely represents a mechanism to block polyspermy

Calcium Signaling in Reproductive and Biotrophic Interactions (Antoine et al., 2000). These data strongly suggested that Ca2+ signaling observed in vitro might resemble the in vivo situation occurring during double fertilization in flowering plants. However, the isolated gametes in the cell fusion medium could not mimic the intimate cell–cell communication events or the physiological conditions in vivo, therefore the observed Ca2+ signatures in in vitro fertilization systems may not indicate the Ca2+ signaling events occurring in vivo. Using a modified semi-in vivo fertilization system and the improved Ca2+ sensor CerTN-L15, Denninger et al. (2014) successfully visualized the occurrence of cytosolic Ca2+ signals during the entire double fertilization process in Arabidopsis. In both egg and central cells (Figure 2C–2E), a sharp increase in [Ca2+]cyt lasting for about 1 min was associated with pollen tube burst and rapid delivery of sperm cells. This short [Ca2+]cyt transient coincided with egg and central cell deformation (Supplemental Movies 2 and 3) due to the explosive release of pollen tube contents. This finding indicates that mechanosensitive channels (e.g. SACs) may mediate Ca2+ influx from the extracellular space. Notably, these [Ca2+]cyt elevations were not observed when pollen tubes randomly deformed female gametes during growth around the egg apparatus. Both pollen tube and receptive synergid cells release a high concentration of Ca2+ during their rupture (Ngo et al., 2014), which likely provides a Ca2+ environment for subsequent gamete fusion events and is the source of Ca2+ influx for Ca2+ signaling. A second extended [Ca2+]cyt transient with more gradual rise and fall times lasting on average for 3.1 ± 0.66 min was detected only in egg cells (Figure 2E), and was correlated with gamete fusion and successful fertilization. A parallel study by Hamamura et al. (2014) revealed similar [Ca2+]cyt signatures using a different Ca2+ sensor, YC3.60 (see also above). The first [Ca2+]cyt peak of 2.8 ± 1.0 min duration occurred simultaneously in the entire egg cell and corresponded to the timing of pollen tube discharge. Meanwhile, a similar Ca2+ spike also occurred in the central cell. The second peak in the egg cell of 4.7 ± 1.2 min duration correlated with egg cell fertilization. It started at the fusion site and then spread over the entire cell. A second minor Ca2+ spike was observed in the central cell with 1.7 ± 0.7 min duration, which was not correlated with fertilization events. In summary, these two novel studies revealed highly specific [Ca2+]cyt signatures regulated by timing and behavior in each female cell type that are different from those observed in vitro or in animal systems. The methodology developed can now be used to extend our knowledge on Ca2+ signaling, for example, in gamete activation and the prevention of polyspermy (entry of multiple sperm cells in female gametes). Egg and central cell activation induces many cellular processes including protein synthesis and the cell cycle. The Ca2+ theory of gamete activation proposes that an increase in concentration of [Ca2+]cyt is responsible, at least in part, for triggering the initiation of these processes in the female gametes. In animal oocytes, this typically involves a wave or waves of Ca2+ release from internal stores and a transient depolarization of the membrane potential. In Arabidopsis, the first sharp increase in [Ca2+]cyt in egg cells and central cells may activate both female gametes and facilitate exocytosis of, for example, vesicles stored in the egg cell contain-

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ing small proteins such as EC1 to activate sperm cells for subsequent double fertilization (Sprunck et al., 2012). The subsequent gamete adhesion step did not induce any visible cytosolic effects in the egg cell, whereas the second extended [Ca2+]cyt transient occurring about 2–5 min after the first elevation was associated with gamete fusion (plasmogamy). Although its duration (3–5 min) is much shorter compared with in vitro fertilization experiments in maize, where monotonic and homogeneous [Ca2+]cyt elevations last up to 30 min, they share common features as they are induced after fusion, spread from the fusion site to the entire cell, and may involve similar mechanisms, for example, a sperm-dependent Gd3+-insensitive Ca2+ influx at the fusion point and a subsequent Gd3+-sensitive influx that is required for sperm incorporation (Antoine et al., 2001). Sperm cell incorporation may also depend on Ca2+regulated cytoskeleton reorganization that enables the delivery of the sperm nucleus to the egg cell nucleus for subsequent karyogamy. Maize egg cells fertilized in vitro do not fuse with additional sperm cells (Faure et al., 1994), suggesting the existence of a block to polyspermy. It is unclear whether a fast block to polyspermy occurs in plants, as in animal species. However, this may be established in the first minute after fusion, for example, by generation of pectin and primary cell wall material after release of homogalacturonic vesicles stored in the egg cell. This was indicated by maize in vitro fertilization studies showing the formation of cell wall material within 30 s after gamete fusion (Kranz et al., 1995). Notably, Ca2+ influx by Ca2+ ionophore treatment of unfertilized egg cell was reported to be sufficient for cell wall deposition without sperm cell activity (Antoine et al., 2000), indicating direct and efficient regulation of Ca2+ signaling to block polyspermy and probably to initiate very early embryogenesis. Moreover, cytosolic Ca2+-dependent cytoskeleton reorganization and vacuole fragmentation may facilitate the subsequent establishment of zygote polarization. In summary, it is very likely that Ca2+ spiking occurring at defined stages during the double fertilization process triggers processes such as gamete activation, a fast block to polyspermy, and induction of zygote development in plants. Studies of mammalian egg activation implicate sperm-specific PLCz, which is introduced into the oocyte following membrane fusion, as the responsible factor for oocyte activation. PLCz can induce Ca2+ oscillations after its encoding RNA or recombinant PLCz is injected into mouse eggs (Miyazaki and Ito, 2006; Kashir et al., 2010). In maize in vitro fertilization assays, Antoine et al. (2001) revealed a sperm-dependent Gd3+-insensitive Ca2+ influx at the fusion point, leaving open the intriguing possibility that this sperm-dependent initiating [Ca2+]cyt elevation also involves maize sperm PLCz. PLCz may subsequently trigger Ca2+-induced Ca2+ release (Uhleń and Fritz, 2010), and this could be (partly) responsible for the spread of the Ca2+ transient from the fusion point to the whole cell. With reference to well-characterized animal model systems, sperm cell and egg cell transcriptome studies will facilitate the identification of key factors involved in egg activation. Moreover, combination of loss-of-function mutants with the above observation system and the application of inhibitory drugs in future studies will greatly accelerate the identification of the key molecular players in the related Ca2+signaling pathways. Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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ROLE OF CALCIUM IN PLANT INTERACTIONS WITH BIOTROPHIC FILAMENTOUS MICROBES Pollen tube invasion appears similar to infection of plant tissue by biotrophic fungi and oomycetes. Extensive communication between the intruder and the receptive cells take place finally allowing access into a cell or tissue. During infection, the invaded cell restructures and polarizes toward the point of invasion in defense or polarizes inward to accommodate the microbe in compatible interactions (Figure 2F). The first genetic evidence for common molecular mechanisms that regulate both pollen tube invasion and compatibility with a biotrophic fungal pathogen stems from analysis of feronia mutants that are not only perturbed in pollen tube reception but also in cell entry and reproductive success by the ascomycetous fungus Erysiphe orontii (Kessler et al., 2010). Plant tissue colonization by biotrophic filamentous microbes follows a similar sequence of events independent of whether the outcome of the interaction is symbiotic or pathogenic (reviewed in Parniske, 2000). Prior to colonization, microbe-released mobile ligands termed microbe-associated molecular patterns (MAMPs) are recognized by extracellular plant receptor kinases, which trigger early immune or symbiotic responses (Antolıń-Llovera et al., 2012). Then the microbes attach to the plant surface and subsequently breach the cell wall to enter the tissue. Finally, the microbes insert feeding structures into plant cells, which are more simple haustoria of biotrophic pathogens and more complex highly branched arbuscules of arbuscular mycorrhizal (AM) fungi. In both pathogenic and symbiotic interactions, intracellular infection leads to drastic subcellular rearrangement and the formation of a new plant-derived membrane domain, the peri-haustorial or peri-arbuscular membrane that surrounds the microbial structure (reviewed by Yi and Valent, 2013; Gutjahr and Parniske, 2013). Ca2+ signaling occurs during root interactions with symbiotic AM fungi and possibly plays an important role in intracellular accommodation of the microbe (Singh and Parniske 2012). However, Ca2+ signaling is also important in innate immune responses that trigger defense against pathogens. How response specificity downstream of Ca2+ fluxes is achieved represents an exiting current research question.

Ca2+ Signaling in Defense Stimulation of plant cells by MAMPs causes dose-dependent rapid increases in (Ca2+)cyt, and different MAMPs induce spatio-temporal Ca2+ patterns with distinct frequency, amplitude, and duration (Blume et al., 2000; Navazio et al., 2007; Ranf et al., 2011, 2012; Seybold et al., 2014; see also Figures 1B, 2F, and 2G). It is currently unclear whether the Ca2+ pattern conveys information and thus determines specific downstream responses or whether Ca2+ influx simply acts as a binary switch that activates Ca2+ binding proteins when a threshold concentration of Ca2+ is reached, while specificity is provided by parallel mechanisms (Seybold et al., 2014). CDPKs emerge as crucial Ca2+ sensors in plant immunity (Romeis and Herde, 2014). For example, treatment of tobacco leaves with the fungal elicitor Avr9 led to rapid activation of CDPK2 (Romeis et al., 2000, 2001). In Arabidopsis, several CDPKs (CDPK4, 5, 6, and 604


11) were stimulated after application of the bacterial MAMP flagellin22 (flg22) and use of Ca2+-channel inhibitors showed that this effect is Ca2+ dependent (Boudsocq et al., 2010). A large overlap between transcriptional responses to overexpression of dominant active versions of CPDK5 and 11 and to treatment with chitin octamers indicated that these CDPKs might also function in innate immune responses to classical fungal MAMPs (Boudsocq et al., 2010). While Ca2+ influx in defense is thought to activate several different downstream responses, it also plays an important role is the promotion of ROS bursts (Ranf et al., 2011; Segonzac et al., 2011; Frei dit Frey et al., 2012; Seybold et al., 2014). ROS burst is often associated with a hypersensitive response leading to cell death, thus inhibiting accommodation of a biotrophic pathogen inside the cell (Ma and Berkowitz 2007). Accumulating evidence suggests a direct role of CDPKs in the generation of ROS bursts during immune responses via activation of NADPH oxidases of the RBOH family, the main players in defense-related ROS production (see also above). These become phosphorylated at serine residues at their amino terminus in a Ca2+-dependent manner (Kobayashi et al., 2007, 2012; Dubiella et al., 2013). Overexpression of a dominant active version of potato CDPK5 in tobacco leaves causes ROS production and increased resistance against the biotrophic oomycete Phytophtora infestans (Kobayashi et al., 2007, 2012). The ability of Arabidopsis CDPK5 to be activated by flg22 and ROS, the fact that it phosphorylates RBOHD and that both cdpk5 and rbohd mutants are defective in systemic defense induction suggested that the CDPK5/RBOHD pair are involved in rapid cell-to-cell long-distance signaling (Dubiella et al., 2013). This is likely based on a self-propagating wave of Ca2+ and ROS in which Ca2+ influx would activate CDPK5, which would in turn activate RBOHD followed by production of ROS. ROS would apoplastically diffuse to the next cell, trigger Ca2+ influx, which would again activate CDPK5 (Romeis and Herde, 2014).

Ca2+ Signaling in Compatible Pathogenic Interactions Very little is known about the role of Ca2+ signaling in mediating compatibility in interactions with biotrophic pathogens. To our knowledge, a description of Ca2+ signatures during successful colonization by biotrophic pathogens is not available. However, the compatibility factor MLO, needed for successful colonization of cereal leaves by adapted pathovars of the powdery mildew fungus Erysiphe graminis requires Ca2+-dependent CaM binding to suppress defense (Kim et al., 2002). Furthermore, it emerges that CDPK family members play a role in compatibility in biotrophic pathogen interactions providing indirect evidence for an involvement of Ca2+ in accommodation of pathogens in host cells. In rice, for example, overexpression of CDPK12 led to increased susceptibility to the blast fungus Magnaporte oryzae (Asano et al., 2012). More impressively, in the barley mlo mutant, which is normally resistant to the adapted powdery mildew fungus, overexpression of constitutive active CDPK3 in leaf cells restored host cell entry (Freymark et al., 2007). Interestingly, gain of CDPK3 function also compromised penetration resistance to a non-adapted wheat powdery mildew fungus in barley wild-type (Freymark et al., 2007) suggesting that CDPK3 negatively regulates defense. This might also be the case for Medicago CDPK1, which is required for root colonization by

Calcium Signaling in Reproductive and Biotrophic Interactions AM fungi and suppression of defense gene induction (Ivashuta et al., 2005). It appears that different CDPK family members play opposite roles in defense and compatibility (Freymark et al., 2007).

NUCLEAR Ca2+ SIGNALING IN SYMBIOTIC INTERACTIONS The major signaling pathway controlling root colonization by symbiotic AM fungi and rhizobia includes intracellular Ca2+ oscillations (Figure 1B). Therefore, the role of Ca2+ signaling in compatible plant–fungus interactions is best understood in symbiosis (Singh and Parniske, 2012; Charpentier and Oldroyd, 2013). Cytoplasmic and nuclear localized cameleon Ca2+ sensors indicated that these Ca2+ oscillations are limited to the nucleus and the perinuclear cytoplasm (Miwa et al., 2006; Sieberer et al., 2009; Capoen et al., 2011). Ca2+ spiking occurs during several phases of root colonization by AM fungi (Gutjahr and Parniske, 2013). A rather chaotic, low-frequency Ca2+ spiking is induced in epidermal cells after treatment with chitin tetra- and pentamers present in the exudates of germinating fungal spores (Kosuta et al., 2008; Chabaud et al., 2011, Genre et al., 2013) and under hyphopodia, which are hyphal attachment structures formed by the fungus at the root surface (Chabaud et al., 2011). Regular high-frequency spiking accompanies fungal passage through epidermal or cortical cells (Sieberer et al., 2012). The meaning of two different Ca2+ signatures is yet unclear but it has been suggested that low-frequency Ca2+ spiking is associated with reversible priming of the cell for infection and high-frequency Ca2+ spiking with a commitment to infection upon fungal entry (Sieberer et al., 2012). In fact, hyphopodium attachment to the root surface triggers subcellular rearrangements in underlying epidermal cells that lead to the formation of a membrane-bound cytoplasmic bridge called the pre-penetration apparatus (PPA) through the vacuole that is lined by cytoskeleton, ER, and cytoplasm and which guides the fungal hypha through the cell (Genre et al., 2005). Lowfrequency Ca2+ spiking occurs during the first phase of subcellular rearrangement. Fungal cell entry is thereafter associated with a switch to higher-frequency spiking (Sieberer et al., 2012). While an epidermal cell is traversed, the two underlying cortical cells start rearranging and show lowfrequency Ca2+ spiking. Only one of the two cells becomes colonized and switches to high-frequency Ca2+ spiking, while the other one ceases spiking and goes back toward its original state (Sieberer et al., 2012). Ca2+ spiking during infection might be triggered by similar chitin-based signaling molecules that trigger Ca2+ spiking prior to infection and possibly a certain localized concentration of signaling molecules is required to induce high-frequency spiking. The observation of different Ca2+ spiking patterns occurring simultaneously in neighboring cells shows that Ca2+ spiking responses to fungal colonization are cell autonomous. However, it is unclear whether the cell showing lowfrequency spiking reacts to a low concentration of diffusing signaling molecules released by the fungus or whether it is somehow influenced by the neighboring cell that shows high-frequency spiking. Another open question relates to whether low spiking frequencies still encode information.

Molecular Plant

Generation of Nuclear Ca2+ Spiking in Root Symbiosis Ca2+ spiking is triggered after perception of chitin-related signaling molecules by LysM-domain containing receptor-like kinases (Op den Camp et al., 2011) that act in concert with SYMBIOSIS receptor-like kinase SYMRK/DMI1 (Ried et al., 2014). Genetic approaches in the legumes Lotus japonicus and Medicago truncatula have identified several components localized in the nuclear membrane that are required for AM colonization and the generation of nuclear Ca2+ spiking (Singh and Parniske, 2012, Charpentier and Oldroyd, 2013). These include the ion channels LjCASTOR and LjPOLLUX/MtDMI1, which predominantly transport K+ and localize to the nuclear membrane (Peiter et al., 2007; Charpentier et al., 2008; Venkateshwaran et al., 2012). DMI1 localizes to both nuclear membranes, but preferentially to the inner membrane (Capoen et al., 2011). Furthermore, a SERCA type ATPase MCA8 has been implicated in the generation of Ca2+ spiking and localizes to both nuclear membranes (Capoen et al., 2011). The localization of proteins required for the generation of Ca2+ spiking together with the observation of Ca2+ peaks predominantly in the periphery of the nucleus (Sieberer et al., 2009; Capoen et al., 2011) suggests that Ca2+ is stored in the peri-nuclear lumen and possibly the associated ER. A Ca2+ channel that generates the Ca2+ spike has not been identified so far but mathematical models predict a requirement of at least three components for the generation of Ca2+ spikes: (i) the potassium channel DMI1 (or CASTOR and POLLUX), which causes a potassium current that leads to changes in membrane potential, which (ii) leads to opening of a putative voltage-gated Ca2+ channel that generates Ca2+ spikes. An ATPase then (iii) pumps Ca2+ back into the store (Granqvist et al., 2012). Furthermore, three components of the nuclear core complex NUP85/NUP133 and NENA are perturbed in Ca2+ spiking and symbiosis development (Kanamori et al., 2006; Saito et al., 2007; Groth et al., 2010) and thus have been reported to be required for transport of proteins such as DMI1 to the inner nuclear membrane (Parniske, 2008).

Decoding of Nuclear Ca2+ Spiking by CCamK It is widely accepted that nuclear Ca2+ spiking is decoded by the nuclear localized chimeric Ca2+- and CaM-dependent kinase CCamK/DMI3 (Singh and Parniske, 2012). Initially, CCamK was found to be expressed in developing anthers of lily (Patil et al., 1995). In AM symbiosis, CCamK/DMI3 is required for intracellular accommodation in the epidermis and cortex (Levy et al., 2004; Mitra et al., 2004; Kistner et al., 2005) and the formation of epidermal PPAs (Genre et al., 2005). Overexpression of a dominant active version of CCamK in the absence of a fungus leads to spontaneous cytoplasmic aggregations that resemble PPAs (Takeda et al., 2012). This indicates that CCamK is necessary and sufficient for PPA formation and that the main function of upstream players involved in the generation of Ca2+ spiking is the activation of CCamK. Consistently, AM colonization can be restored by overexpressing a dominant active CCamK version in otherwise AM-deficient symrk, castor, pollux, and nup85 mutants (Hayashi et al., 2010). CCamK/DMI3 contains a CaM binding domain and three carboxyterminal EF hands (Levy et al., 2004; Mitra et al., 2004). Its amino terminus resembles CaMKII, which can decode Ca2+ spiking frequencies in nerve-cell signal transduction (de Koninck and Schulman, 1998). It has long been assumed that Molecular Plant 8, 595–611, April 2015 ª The Author 2015.

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Ca2+ binding to the EF hands leads to autophosphorylation at threonine 265 in Lotus (271 in Medicago), which activates CCamK/DMI3 (Gleason et al., 2006; Shimoda et al., 2012). However, a recent study suggests that autophosphorylation promoted by Ca2+ binding to the EF hands at basal Ca2+ concentrations stabilizes the inactive state of the protein (Miller et al., 2013). High Ca2+ concentrations during spiking induce CaM binding to CCamK/DMI3, which supersedes autophosphorylation and activates the protein (Miller et al., 2013). Activated CCamK interacts with and phosphorylates the transcription factor CYCLOPS, which activates the promoter of the Nod factor-induced gene NODULE INCEPTION (NIN) (Singh et al., 2014). Since both CCamK and CYCLOPS are required for AM formation (Yano et al., 2008) and auto-active CCamK can spontaneously induce genes that are specifically induced during AM (Takeda et al., 2012), it can be expected that the CCamK/ CYLOPS complex also regulates AM-induced genes.

CONCLUSIONS AND OUTLOOK In this review, we focused on Ca2+ signaling induced after physical interaction between cells, especially on recent research reporting Ca2+ signatures and their periodicity in reproductive and fungal interactions. Initially, we aimed to compare Ca2+ signatures and the associated molecular mechanisms leading to release of Ca2+ from internal and external stores and the enzymatic machinery involved in decoding the various types of transient Ca2+ elevations. However, this approach appeared quite challenging as Ca2+ signatures and periodicity are highly diverse and partly chaotic depending on the inducing stimulus and interacting cell types. Another difficulty is the limited knowledge about the decoding machinery. Moreover, studies in legumes, for example, showed that both bacterial- and fungal-induced Ca2+ responses are dependent on the same proteins, DMI1 and DMI2, but involve different Ca2+ patterns (Kosuta et al., 2008). Regular Ca2+ spiking patterns were observed after bacterial infection, while infection by mycorrhizal fungi also includes more irregular and chaotic patterns. Thus, Ca2+ signaling during fungal invasion might not only be required for regulation of gene expression as previously proposed. It is a valid hypothesis that, similar to its involvement during pollen tube growth or zygote polarization (Bothwell et al., 2008), Ca2+ signaling might also be involved in fast processes such as regulating cytoskeletal rearrangement, vacuolar fragmentation, vesicle transport, and fusion for synthesis of the perifungal membrane during intracellular accommodation of the filamentous microbe. Thus, the direct connection between Ca2+ signaling and these aspects of cellular rearrangement is subject to future investigation as the only experimental evidence is the absence of PPA formation in mutants perturbed in the generation of Ca2+ spiking. In summary, stimulation of plant cells during reproduction and fungal interactions causes a rapid increase in Ca2+, but spatio-temporal Ca2+ signatures significantly differ in frequency, amplitude, duration, and their subcellular location and thus cannot be correlated with biological processes such as compatible interactions, cytoskeletal stability, or cell death. A very obvious difference with Ca2+ signaling is the finding that Ca2+ spiking involving reproductive cells occurs cytoplasmically, while symbiotic interactions are also associated with nuclear Ca2+ oscillations. Cytoplasmic Ca2+ elevations strongly increase 606


the availability of [Ca2+]cyt and thus lead to fast responses such as the modulation of Ca2+ transport via activation of Ca2+ pumps and inactivation of Ca2+ channels, respectively, vesicle release and cytoskeletal rearrangement. These processes are correlated in reproduction with fast and flexible growth behavior and fertilization mechanisms, which occur within a few minutes (Dresselhaus and Franklin-Tong, 2013). Slow responses result in the alteration of gene expression and thus likely play the major role after nuclear spiking. Moreover, Ca2+ spiking during reproduction involves transient cytoplasmic Ca2+elevations in the region below the surface of interacting cells. This indicates Ca2+ influx but it cannot be excluded that Ca2+ is also released from internal stores especially from the ER in this region. Oscillation at guard cells, for example, was recently suggested to depend on both external and internal Ca2+ stores (Thor and Peiter, 2014), which might be a more general phenomenon and always occur simultaneously. As shown in Figure 1, both cytoplasmic and nuclear Ca2+ transients depend on the activation of surface receptors such as FER, LRE, and PrpS during reproduction (Figure 1A), or FER, FLS2, BAK1, Cf9, CERK1, and CEBIP in pathogenic and LysM RLK and SYMRK in symbiotic interactions (Figure 1B). Stimulating ligands are either small extracellular proteins/ peptides or chitin oligomers. Receptor activation thereafter results in opening of channels including SACs, CNGCs, and GLRs, but the extent to which the various channels contribute to Ca2+ elevations and their regulation after receptor activation is largely unclear and requires extensive future research efforts. Oscillations induced, for example, by FLG22 in guard cells were shown to be abolished by EGTA and other drugs, but not by inhibitors of CNGCs and GLRs (Thor and Peiter, 2014) indicating the presence of other not yet identified major Ca2+ channels. Another interesting observation is the finding that Ca2+ influx also plays an important role in the promotion of ROS burst, which was previously associated with a hypersensitive response leading to cell death in defense reactions. This would ultimately inhibit the accommodation of a biotrophic pathogen inside the cell (Ma and Berkowitz 2007). ROS production in synergid cells may also be required for cell death, but this remains to be shown during further experimentation. First studies have indicated that high amplitudes of Ca2+ spikes over a longer time period are correlated to cell death of both pollen tubes and synergid cells (Denninger et al., 2014; Ngo et al., 2014) and it will now be interesting to quantify the correlation of ROS production with the death of reproductive cells. Systematic approaches including phospho-proteomics are now necessary to investigate how Ca2+ signatures are interpreted by the battery of sensor and relay proteins expressed in each cell. CDPKs, for example, which are also involved in the generation of ROS via phosphorylation of NADPH oxidases are rapidly activated by Ca2+ after immune stimulation and appear as central downstream regulators. However, their role in reproduction and symbiotic interactions is almost entirely unclear. In conclusion, the strong overlap of reproductive and defense signaling mechanisms including the Ca2+ signaling network will help us to understand the role of Ca2+ signaling and inspiration can be gleaned from the respective other process.

Calcium Signaling in Reproductive and Biotrophic Interactions SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

FUNDING Work in the C.G.’s and T.D.’s laboratories is supported by grants from the German Research Council (DFG) via the Collaborative Research Center SFB924. A.B. is funded by the Collaborative Research Center SFB960 and J.C. by the Priority Program SSP1365/2 of the DFG to T.D.

Molecular Plant

Boudsocq, M., Willmann, M.R., McCormack, M., Lee, H., Shan, L., He, P., Bush, J., Cheng, S.-H., and Sheen, J. (2010). Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464: 418–422. Brewbaker, J.L., and Kwack, B.H. (1963). The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50: 859–865.

No conflict of interest declared.

Capoen, W., Sun, J., Wysham, D., Otegui, M.S., Venkateshwaran, M., Hirsch, S., Miwa, H., Downie, J.A., Morris, R.J., Ane´, J.-M., et al. (2011). Nuclear membranes control symbiotic calcium signaling of legumes. Proc. Natl. Acad. Sci. USA 108:14348–14353.

Received: December 8, 2014 Revised: January 18, 2015 Accepted: January 20, 2015 Published: February 4, 2015

Chabaud, M., Genre, A., Sieberer, B.J., Faccio, A., Fournier, J., Novero, M., Barker, D.G., and Bonfante, P. (2011). Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis. New Phytol. 189:347–355.

ACKNOWLEDGMENTS

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611

Calcium signaling in plants.

Extracellular Recognition of Oomycetes during Biotrophic Infection of Plants.

Editorial: Biotrophic Plant-Microbe Interactions.

Calcium signaling and biotic defense responses in plants.

Tissue magnesium and calcium affect arbuscular mycorrhiza development and fungal reproduction.

Competence and regulatory interactions during regeneration in plants.

Nuclear proton dynamics and interactions with calcium signaling.

Matrix metalloproteinases operate redundantly in Arabidopsis immunity against necrotrophic and biotrophic fungal pathogens.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling.

Host-pathogen interactions in plants. Plants, when exposed to oligosaccharides of fungal origin, defend themselves by accumulating antibiotics.

Genomics and evolutionary aspect of calcium signaling event in calmodulin and calmodulin-like proteins in plants.

Components of the calcium-calcineurin signaling pathway in fungal cells and their potential as antifungal targets.

Cell interactions in plants.

Histone acetylation in fungal pathogens of plants.

Emerging Trends in Molecular Interactions between Plants and the Broad Host Range Fungal Pathogens Botrytis cinerea and Sclerotinia sclerotiorum.

Tetrapyrrole Signaling in Plants.

Introduction: signaling in plants.

Cell interactions and cell signaling during hematopoietic development.

Calcium imaging perspectives in plants.

Reduction of Growth and Reproduction of the Biotrophic Fungus Blumeria graminis in the Presence of a Necrotrophic Pathogen.

Estradiol Membrane-Initiated Signaling and Female Reproduction.

DNA fingerprinting of Peronospora parasitica, a biotrophic fungal pathogen of crucifers.

Dimorphism in fungal pathogens of mammals, plants, and insects.

Plant reproduction and environmental noise: how do plants do it?