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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. [ABSTRACT FROM AUTHOR]

Summary: Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non‐specific pharmacological approaches. We therefore developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the 'cAMP‐sponge' (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with wild‐type (WT) plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24 h post‐infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium‐calmodulin‐binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis‐regulation of AtSR1 activity, affected the speed and strength of the immune response. Significance Statement: This study introduces the use of the cAMP‐sponge as a genetic tool to investigate the role of cAMP in plant immune responses, revealing the complexity of the signal network involved in the establishment of plant resistance against an avirulent strain of Pseudomonas syringae pv. tomato. The decrease in free cAMP content, altering the timing and levels of cytosolic calcium and hydrogen peroxide signals upon pathogen perception, affected the speed and strength of the immune response. [ABSTRACT FROM AUTHOR]

Ca2+ signals are transient, hence, upon a stimulus-induced increase in cytosolic Ca2+ concentration, cells have to reestablish 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. [ABSTRACT FROM AUTHOR]

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine≅phosphatidylethanolamine≅0. Acidic phospholipids increased Vmax-Ca2+ and lowered the value of K0.5-Ca2+ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K0.5-Ca2+ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Δ74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein. [ABSTRACT FROM AUTHOR]

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca2+-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K0.5 value for the complex of 15 ± 1 µg ml−1, which is unaffected by free [Ca2+]. Heparin increases Vmax up to 3-fold while it does not significantly affect the apparent Km for free Ca2+ and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain (Δ74–ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin–agarose gel show that heparin directly interacts with ACA8. Binding to the heparin–agarose gel occurs also with a peptide reproducing ACA8 sequence 1M-I116. Several single-point mutations within ACA8 sequence A56–T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca2+-ATPase and its animal counterpart, which is inhibited by heparin. [ABSTRACT FROM PUBLISHER]

Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At-ACA8, the first cloned A. thaliana PM Ca2+-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At-ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide 41I-T63 had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µM free Ca2+ about 25 nM], thus localizing At-ACA8 CaM-binding site within this sequence. The interaction of At-ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca2+-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca2+-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca2+ concentration, suggesting that enzyme conformation is affected by Ca2+. Binding of CaM isoforms to At-ACA8 N-terminus was affected differently by free Ca2+ concentration, suggesting that plant CaMs may have different affinities for Ca2+. [ABSTRACT FROM AUTHOR]

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (EC 3.6.3.6) (Morandini P, Valera M, Albumi C, Bonza MC, Giacometti S, Ravera G, Murgia I, Soave C & De Michelis MI (2002) Plant J 31, 487–497). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H+-ATPase. PPI1-induced stimulation of H+-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H+-ATPase. These results indicate that PPI1 can bind the H+-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action. [ABSTRACT FROM AUTHOR]

Type IIB Ca2+-ATPases have a terminal auto-inhibitory, domain the action of which is suppressed by calmodulin (CaM) binding. Here, we show that a peptide (6His-1M-I116) corresponding to the first 116 aminoacids (aa) of At-ACA8, the first cloned isoform of Arabidopsis thaliana plasma membrane Ca2+-ATPase, inhibits the activity of the enzyme deprived of the N-terminus by controlled trypsin treatment 10-fold more efficiently than a peptide (41I-T63) corresponding only to the CaM-binding site. A peptide (268E-W348) corresponding to 81 aa of the small cytoplasmic loop of At-ACA8 binds peptide 6His-1M-I116 immobilized on Ni–NTA agarose. Peptide 268E-W348 stimulates Ca2+-ATPase activity. Its effect is not additive with that of CaM and is suppressed by tryptic cleavage of the N-terminus. These results provide the first functional identification of a site of intramolecular interaction with the terminal auto-inhibitory domain of type IIB Ca2+-ATPases. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

ACA8 is a type 2B Ca2+-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 Glu252 to Asn345 sequence. Mutation to Ala of any of six tested acidic residues (Glu252, Asp273, Asp291, Asp303, Glu302, or Asp332) 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 wildtype 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 Ca2+-ATPase plays a role in the attainment of the auto-inhibited state. [ABSTRACT FROM AUTHOR]

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 At GLR3.3 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 At GLR3.3 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. [Display omitted] • Plant glutamate receptor-like channel At GLR3.3 is gated by exogenous amino acids • At GLR3.3 ligand-binding domain is essential for channel activation • Leaf injury and osmotic stress induce L-Glu systemic elevation activating At GLR3.3 • Local activation of GLRs does not trigger long-distance Ca2+ waves In Arabidopsis thaliana , mechanical stresses induce long-distance Ca2+ signals mediated by the glutamate receptor-like 3.3 channel. Grenzi et al. show that the stress-induced systemic increase of apoplastic L-glutamate, in a submillimolar range of concentrations, gates the At GLR3.3 through its ligand-binding domain. [ABSTRACT FROM AUTHOR]