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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]

Abstract: Plant auto-inhibited Ca2+-ATPase 8 (ACA8) and animal plasma membrane Ca2+-ATPase 4b (PMCA4b) are representatives of plant and animal 2B P-type ATPases with a regulatory auto-inhibitory domain localized at the N- and C-terminus, respectively. To check whether the regulatory domain works independently of its terminal localization and if auto-inhibitory domains of different organisms are interchangeable, a mutant in which the N-terminus of ACA8 is repositioned at the C-terminus and chimeras in which PMCA4b C-terminus is fused to the N- or C-terminus of ACA8 were analysed in the yeast mutant K616 devoid of endogenous Ca2+-ATPases. Results show that the regulatory function of the terminal domain is independent from its position in ACA8 and that the regulatory domain belonging to PMCA4b is able to at least partially auto-inhibit ACA8. [ABSTRACT FROM AUTHOR]

In plant Ca2+ pumps belonging to the P2B subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CAM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg43 to Lys68) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix. [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]

The plasma membrane Ca 2 + ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca 2 + ), 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 Ca 2 + ), Ca 2 + -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 Ca 2 + in the activated state, and the autoinhibition mechanism in its resting state. [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]

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 EFhands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two lowaffinity 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. [ABSTRACT FROM AUTHOR]

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.