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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., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ca 2+ 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 Ca 2+ signatures and their encoding and decoding machinery. Such studies have frequently focused on Ca 2+ alone and our understanding of how Ca 2+ 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 Ca 2+ 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-Ca 2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca 2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca 2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca 2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity., (© 2018 American Society of Plant Biologists. All rights reserved.)

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 (Ca 2+ ) dynamics and changes of the glutathione redox status taking place in plant organs during plant interaction with the HR-inducing bacteria Pseudomonas syringae (PstAvrB).

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2+ )-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2+ 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 Ca 2+ and Mg 2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2+ /Mg 2+ mixed binding sites and two low-affinity Ca 2+ -specific sites. Binding of Ca 2+ 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 Ca 2+ or Mg 2+ , 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 Ca 2+ -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 Ca 2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2+ pumps., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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]

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]