The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel.

Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.

The signatures of organellar calcium.

Recent insights about the transport mechanisms involved in the in and out of calcium ions in plant organelles, and their role in the regulation of cytosolic calcium homeostasis in different signaling pathways.

Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants.

Calcium ions (Ca2+) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca2+ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca2+ store that actively accumulates Ca2+ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca2+ indicators, specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca2+ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified (1) the time required by the ER to import Ca2+ from the cytosol into the lumen and (2) the time required to observe a cytosolic Ca2+ increase upon the pharmacological inhibition of the ER-localized P-Type IIA Ca2+-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca2+ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca2+ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca2+ homeostasis and signaling.

Structural insights into long-distance signal transduction pathways mediated by plant glutamate receptor-like channels.

In recent years, studies have shed light on the physiological role of plant glutamate receptor-like channels (GLRs). However, the mechanism by which these channels are activated, and in particular, what is the physiological role of their binding to amino acids, remains elusive. The first direct biochemical demonstration that the Arabidopsis thaliana GLR3.3 isoform binds glutamate and other amino acids in a low micromolar range of concentrations was reported only recently. The first crystal structures of the ligand-binding domains of AtGLR3.3 and AtGLR3.2 isoforms also have been released. We foresee that these new experimental pieces of evidence provide the basis for a better understanding of how GLRs are activated and modulated in different physiological responses.

Cellular Ca2+ Signals Generate Defined pH Signatures in Plants.

Ca2+ play a key role in cell signaling across organisms. The question of how a simple ion can mediate specific outcomes has spurred research into the role of Ca2+ signatures and their encoding and decoding machinery. Such studies have frequently focused on Ca2+ alone and our understanding of how Ca2+ signaling is integrated with other responses is poor. Using in vivo imaging with different genetically encoded fluorescent sensors in Arabidopsis (Arabidopsis thaliana) cells, we show that Ca2+ transients do not occur in isolation but are accompanied by pH changes in the cytosol. We estimate the degree of cytosolic acidification at up to 0.25 pH units in response to external ATP in seedling root tips. We validated this pH-Ca2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity.

MIZ1 regulates ECA1 to generate a slow, long-distance phloem-transmitted Ca2+ signal essential for root water tracking in Arabidopsis.

Ever since Darwin postulated that the tip of the root is sensitive to moisture differences and that it "transmits an influence to the upper adjoining part, which bends towards the source of moisture" [Darwin C, Darwin F (1880) The Power of Movement in Plants, pp 572-574], the signal underlying this tropic response has remained elusive. Using the FRET-based Cameleon Ca2+ sensor in planta, we show that a water potential gradient applied across the root tip generates a slow, long-distance asymmetric cytosolic Ca2+ signal in the phloem, which peaks at the elongation zone, where it is dispersed laterally and asymmetrically to peripheral cells, where cell elongation occurs. In addition, the MIZ1 protein, whose biochemical function is unknown but is required for root curvature toward water, is indispensable for generating the slow, long-distance Ca2+ signal. Furthermore, biochemical and genetic manipulations that elevate cytosolic Ca2+ levels, including mutants of the endoplasmic reticulum (ER) Ca2+-ATPase isoform ECA1, enhance root curvature toward water. Finally, coimmunoprecipitation of plant proteins and functional complementation assays in yeast cells revealed that MIZ1 directly binds to ECA1 and inhibits its activity. We suggest that the inhibition of ECA1 by MIZ1 changes the balance between cytosolic Ca2+ influx and efflux and generates the cytosolic Ca2+ signal required for water tracking.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.

The ataxia related G1107D mutation of the plasma membrane Ca2+ ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

The plasma membrane Ca2+ ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca2+), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca2+), Ca2+-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca2+ in the activated state, and the autoinhibition mechanism in its resting state.

Ca2+-dependent phosphoregulation of the plasma membrane Ca2+-ATPase ACA8 modulates stimulus-induced calcium signatures.

Ca2+ signals are transient, hence, upon a stimulus-induced increase in cytosolic Ca2+ concentration, cells have to re-establish resting Ca2+ levels. Ca2+ extrusion is operated by a wealth of transporters, such as Ca2+ pumps and Ca2+/H+ antiporters, which often require a rise in Ca2+ concentration to be activated. Here, we report a regulatory fine-tuning mechanism of the Arabidopsis thaliana plasma membrane-localized Ca2+-ATPase isoform ACA8 that is mediated by calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) complexes. We show that two CIPKs (CIPK9 and CIPK14) are able to interact with ACA8 in vivo and phosphorylate it in vitro. Transient co-overexpression of ACA8 with CIPK9 and the plasma membrane Ca2+ sensor CBL1 in tobacco leaf cells influences nuclear Ca2+ dynamics, specifically reducing the height of the second peak of the wound-induced Ca2+ transient. Stimulus-induced Ca2+ transients in mature leaves and seedlings of an aca8 T-DNA insertion line exhibit altered dynamics when compared with the wild type. Altogether our results identify ACA8 as a prominent in vivo regulator of cellular Ca2+ dynamics and reveal the existence of a Ca2+-dependent CBL-CIPK-mediated regulatory feedback mechanism, which crucially functions in the termination of Ca2+ signals.

ACA12 is a deregulated isoform of plasma membrane Ca²âº-ATPase of Arabidopsis thaliana.

Plant auto-inhibited Ca²âº-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca²âº-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues--highly conserved in other ACA isoforms--localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.

Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleon sensor.

In planta, very limited information is available about how the endoplasmic reticulum (ER) contributes to cellular Ca(2+) dynamics and homeostasis. Here, we report the generation of an ER-targeted Cameleon reporter protein suitable for analysis of Ca(2+) accumulation and dynamics in the lumen of the ER in plant cells. Using stably transformed Arabidopsis (Arabidopsis thaliana) plants expressing this reporter protein, we observed a transiently enhanced accumulation of Ca(2+) in the ER in response to stimuli inducing cytosolic Ca(2+) rises in root tip cells. In all experimental conditions, ER Ca(2+) dynamics were substantially different from those monitored in the cytosol. A pharmacological approach enabled us to evaluate the contribution of the different ER-resident Ca(2+)-ATPase classes in the regulation of the ER Ca(2+) homeostasis. Taken together, our results do not provide evidence for a role of the ER as a major source that releases Ca(2+) for stimulus-induced increases in cytosolic Ca(2+) concentration. Instead, our results show that the luminal ER Ca(2+) elevations typically follow cytosolic ones, but with distinct dynamics. These findings suggest fundamental differences for the function of the ER in cellular Ca(2+) homeostasis in plants and animals.

Plant Ca2+-ATPases: From biochemistry to signalling.

Calcium (Ca2+)-ATPases are ATP-dependent enzymes that transport Ca2+ ions against their electrochemical gradient playing the fundamental biological function of keeping the free cytosolic Ca2+ concentration in the submicromolar range to prevent cytotoxic effects. In plants, type IIB autoinhibited Ca2+-ATPases (ACAs) are localised both at the plasma membrane and at the endomembranes including endoplasmic reticulum (ER) and tonoplast and their activity is primarily regulated by Ca2+-dependent mechanisms. Instead, type IIA ER-type Ca2+-ATPases (ECAs) are present mainly at the ER and Golgi Apparatus membranes and are active at resting Ca2+. Whereas research in plants has historically focused on the biochemical characterization of these pumps, more recently the attention has been also addressed on the physiological roles played by the different isoforms. This review aims to highlight the main biochemical properties of both type IIB and type IIA Ca2+ pumps and their involvement in the shaping of cellular Ca2+ dynamics induced by different stimuli.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana.

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca(2+)-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D--and to a lesser extent also mutants S22D and S27D--are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-(1)M-I(116)) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K(D) ≈ 10-fold higher than wild-type ACA8) and S99D (K(D) about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-(1)M-I(116) peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.

Signaling by plant glutamate receptor-like channels: What else!

Plant glutamate receptor-like channels (GLRs) are transmembrane proteins that allow the movement of several ions across membranes. In the model plant Arabidopsis, there are 20 GLR isoforms grouped in three clades and, since their discovery, it was hypothesized that GLRs were mainly involved in signaling processes. Indeed, in the last years, several pieces of evidence demonstrate different signaling roles played by GLRs, related to pollen development, sexual reproduction, chemotaxis, root development, regulation of stomatal aperture, and response to pathogens. Recently, GLRs have gained attention for their role in long-distance electric and calcium signaling. In this review, we resume the evidence about the role of GLRs in signaling processes. This role is mostly linked to the GLRs involvement in the regulation of ion fluxes across membranes and, in particular, of calcium, which represents a key second messenger in plant cell responses to both endogenous and exogenous stimuli.

Single point mutations in the small cytoplasmic loop of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, generate partially deregulated pumps.

ACA8 is a type 2B Ca(2+)-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu(252) to Asn(345) sequence. Mutation to Ala of any of six tested acidic residues (Glu(252), Asp(273), Asp(291), Asp(303), Glu(302), or Asp(332)) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca(2+)-ATPase plays a role in the attainment of the auto-inhibited state.

A functional calcium-transporting ATPase encoded by chlorella viruses.

Calcium-transporting ATPases (Ca(2+) pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca(2+) pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca(2+) pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca(2+) pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a (32)P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca(2+), thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca(2+)-transporting ATPase encoded by a virus.

Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana.

Plants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner.

In Vivo Analysis of Calcium Levels and Glutathione Redox Status in Arabidopsis Epidermal Leaf Cells Infected with the Hypersensitive Response-Inducing Bacteria Pseudomonas syringae pv. tomato AvrB (PstAvrB).

Plants react to the attack of pathogen microorganisms by mounting appropriate and efficient downstream defense responses often involving a form of localized cell death called hypersensitive response (HR).Here we describe an innovative and noninvasive protocol based on in vivo bioimaging technique coupled with utilization of genetically encoded fluorescent sensors that allows to monitor and analyze intracellular calcium (Ca2+) dynamics and changes of the glutathione redox status taking place in plant organs during plant interaction with the HR-inducing bacteria Pseudomonas syringae (PstAvrB).

Unique resistance traits against downy mildew from the center of origin of grapevine (Vitis vinifera).

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.