Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

Calcium and pH patterning at the apical meristem are specifically altered by photoperiodic flower induction in Chenopodium spp.

The apical meristem of the short‐day plant Chenopodium rubrum responds to photoperiodic flower induction with specific changes of pH and Ca²⁺ patterning immediately after the inductive dark span. The red–far‐red reversibility of the pH and Ca²⁺ patterning in response to night break treatments was measured in order to distinguish between the effect of the prolonged dark span per se and the specific effect of photoperiodic flower induction. In addition, the pH and Ca²⁺ patterning in C. rubrum was compared with the long‐day plant Chenopodium murale. The pH was visualized using the fluorescent probe carboxy SNARF‐1. Calcium ion concentrations were studied using a combination of Ca²⁺‐probes Fluo‐3 and Fura Red. It was observed that the specific changes in pH and Ca²⁺ patterning at the apical meristem of C. rubrum were abolished by the red‐light break. This effect was fully reversed with a subsequent single far‐red treatment. These observations infer the influence of phytochrome on both pH and Ca²⁺ patterning. Changes in pH and Ca²⁺ patterning upon flower induction were observed in both long‐day and short‐day plants. These results support the hypothesis that changes of pH and [Ca²⁺] in cells of the apical meristem are part of the pathway in signal transduction triggering flower initiation.     

De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure

Cytosolic Ca²⁺ ([Ca²⁺]_cyt) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca²⁺]_cyt has been shown to regulate stomatal movements, and a defined window of Ca²⁺ oscillation kinetic parameters encodes necessary information for long‐term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca²⁺ signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over‐expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca²⁺‐induced stomatal closure. Cytosolic Ca²⁺ activation of S‐type anion channels, which play a central role in Ca²⁺‐reactive stomatal closure, was normal in the AtGLR3.1 over‐expressing plants. Interestingly, AtGLR3.1 over‐expression did not affect Ca²⁺‐induced Ca²⁺ oscillation kinetics, but resulted in a failure to maintain long‐term ‘Ca²⁺‐programmed’ stomatal closure when Ca²⁺ oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short‐term Ca²⁺‐reactive closure was not affected in AtGLR3.1 over‐expressing plants. In wild‐type plants, the translational inhibitor cyclohexamide partially inhibited Ca²⁺‐programmed stomatal closure induced by experimentally imposed Ca²⁺ oscillations without affecting short‐term Ca²⁺‐reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over‐expressing plants. Our results suggest that over‐expression of AtGLR3.1 impairs Ca²⁺ oscillation‐regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long‐term Ca²⁺‐programmed stomatal closure.     

Ammonium uptake and cellular alkalisation in roots of Arabidopsis thaliana: The involvement of cytoplasmic calcium1

Using roots from Arabidopsis thaliana expressing the recombinant calcium indicator aequorin, we show that NH₃ uptake and alkalisation of plant cells act as a stimulus which induces transient elevations of the cytoplasmic free calcium concentration ([Ca²⁺]_c). The magnitudes of these [Ca²⁺]_c elevations are dependent on the concentration of the membrane permeable form, NH₃, and hence, particularly dependent on the pH in the external medium. EGTA and La³⁺ are able to significantly suppress the [Ca²⁺]_c transients showing that Ca²⁺ influx through the plasma membrane is likely to be involved. Verapamil and nifedipine had no inhibitory effects, which suggests that Ca²⁺ release from internal stores might not contribute significantly to the NH₃‐triggered [Ca²⁺]_c response. Pre‐incubation in l ‐methionine‐dl ‐sulphoximine – an inhibitor of the glutamine synthetase – did not alter the NH₃‐induced [Ca²⁺]_c responses at all. These results are consistent with previous studies where NH₃‐induced changes of cytoplasmic and vacuolar pH were investigated in maize roots. Furthermore, the similarity between the kinetics of NH₃‐driven cellular pH changes demonstrated in previous studies and the [Ca²⁺]_c transients shown here suggests a direct relationship between [Ca²⁺]_c and cellular alkalisation (cytoplasmic pH and/or vacuolar pH). However, the mechanism behind this possible causal relation remains to be elucidated.     

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K⁺ channel activity, cytosolic calcium [Ca²⁺]_cyt and reactive oxygen species (ROS) (H₂O₂ and NO) release, shoot‐to‐root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative β‐galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca²⁺]_cyt, NO and H₂O₂. In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K⁺ channel activity and Vm depolarization, a loss of shoot‐induced root‐Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium‐activated oxidative response and a K⁺‐dependent Vm‐activated jasmonate response associated with the release of VOCs.     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

CML9, an Arabidopsis calmodulin-like protein, contributes to plant innate immunity through a flagellin-dependent signalling pathway

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca²⁺ signals are perceived by various Ca²⁺ sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin‐like (CML) proteins extend the Ca²⁺ toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca²⁺ decoding proteins to plant immunity is emerging, and we report here data on Arabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains of Pseudomonas syringae. Compared to wild‐type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over‐expressing transgenic lines. Collectively, using PAMP treatment and P. syringae strains, we have established that CML9 participates in plant innate immunity.     

Principles of Calcium Signal Generation and Transduction in Plant Cells

Calcium ions exhibit unique properties and a universal ability to transmit diverse signals in plant cells under the primary action of hormones, pathogens, light, gravity, and various abiotic stressors. In the last few years, considerable progress has been achieved in deciphering the mechanisms of Ca²⁺ involvement in the regulation of plant responses. Recent studies revealed the genes encoding Ca²⁺-permeable channels that conduct Ca²⁺ currents across the membranes during the transduction of the Ca²⁺ signal. These proteins comprise the ligand-gated Ca²⁺-permeable channels activated by cyclic nucleotides (CNGC) and amino acids (glutamate receptor-like channels, GLR), the voltage-gated tonoplast channel (two-pore channel, TPC1), mechanosensitive channels (MSL, MCA, OSCA1), and annexins. The role of Ca²⁺-ATPase and Ca²⁺/H⁺-exchangers in the active extrusion of excess cytoplasmic Ca²⁺ into the apoplast or cell organelles was examined in detail. The calmodulins (CaM), CaM-like proteins (CML), Ca²⁺-dependent protein kinases (CDPK), and complexes of calcineurin-B-like proteins (CBL) with CBL-interacting protein kinases (CIPK) were found to produce intricate signaling networks that decode Ca²⁺ signals and elicit plant responses to external stimuli. This review analyzes the data accumulated over the past decade on the principles of formation and propagation of the calcium signal in plant cells.     

A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots

Calcium (Ca²⁺), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca²⁺] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca²⁺ signalling‐related functions. The CWE induces a transient cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY‐2 suspension cultures expressing the Ca²⁺ bioluminescent indicator aequorin. Nuclear Ca²⁺ transients were also observed in tobacco BY‐2 cells. The Ca²⁺ response was more pronounced in roots than in shoots and involved Ca²⁺ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca²⁺ response by staurosporine and the refractory nature of the Ca²⁺ elevation suggest that a receptor may be involved. The CWE does not stimulate H₂O₂ production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca²⁺‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca²⁺ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.     

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat‐induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat‐activated Ca²⁺‐permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole‐cell patch‐clamp technique. The results indicated that A. thaliana cyclic nucleotide‐gated ion channel 6 (CNGC6) mediates heat‐induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat‐shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Chemical signaling under abiotic stress environment in plants

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca²⁺), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Ca²⁺ is one of the very important ubiquitous second messengers in signal transduction pathways and usually its concentration increases in response to the stimuli including stress signals. Many Ca²⁺ sensors detect the Ca²⁺ signals and direct them to downstream signaling pathways by binding and activating diverse targets. cAMP or cGMP protects the cell with ion toxicity. Phosphoinositides are known to be involved both in transmission of signal across the plasma membrane and in intracellular signaling. NO activates various defense genes and acts as a developmental regulator in plants. Sugars affect the expression of many genes involved in photosynthesis, glycolysis, nitrogen metabolism, sucrose and starch metabolism, defense mechanisms and cell cycle regulation. ABA, JA, SA and polyamines are also involved in many stress responses. Cross-talk between these chemical signaling pathways is very common in plant responses to abiotic and bitotic factors. In this article we have described the role of these chemicals in initiating signaling under stress conditions mainly the abiotic stress.Key words: ABA, abiotic stress, Ca²⁺ binding proteins, calcium signaling, cyclic nucleotides, nitric oxide, phosphoinositides signaling, signal transduction, sugar signaling     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.