Maria Cristina Bonza

The plant Ca2+-ATPase repertoire: biochemical features and physiological functions

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.

Analyses of Ca2+ Accumulation and Dynamics in the Endoplasmic Reticulum of Arabidopsis Root Cells Using a Genetically Encoded Cameleon Sensor

Generation of an ER-targeted Cameleon reporter protein enables the analysis of Ca2+ accumulation and dynamics in the lumen of the ER in plant cells. In planta, very limited information is available about how the endoplasmic reticulum (ER) contributes to cellular Ca2+ dynamics and homeostasis. Here, we report the generation of an ER-targeted Cameleon reporter protein suitable for analysis of Ca2+ 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 Ca2+ in the ER in response to stimuli inducing cytosolic Ca2+ rises in root tip cells. In all experimental conditions, ER Ca2+ dynamics were substantially different from those monitored in the cytosol. A pharmacological approach enabled us to evaluate the contribution of the different ER-resident Ca2+-ATPase classes in the regulation of the ER Ca2+ homeostasis. Taken together, our results do not provide evidence for a role of the ER as a major source that releases Ca2+ for stimulus-induced increases in cytosolic Ca2+ concentration. Instead, our results show that the luminal ER Ca2+ elevations typically follow cytosolic ones, but with distinct dynamics. These findings suggest fundamental differences for the function of the ER in cellular Ca2+ homeostasis in plants and animals.

H+/Ca2+ exchange driven by the plasma membrane Ca2+-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification.

The plasma membrane Ca2+‐ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)‐affinity chromatography and reconstituted in proteoliposomes by the freeze‐thaw sonication procedure. The reconstituted enzyme catalyzed CaM‐stimulated 45Ca2+ accumulation and H+ ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca2+ uptake, but it is a primary event linked to Ca2+ uptake in the form of countertransport.

Auto-inhibition of Arabidopsis thaliana plasma membrane Ca2+-ATPase involves an interaction of the N-terminus with the small cytoplasmic loop

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.

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 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 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 Ca2+-ATPase plays a role in the attainment of the auto-inhibited state.

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

The plasma membrane Ca2+-ATPase ACA8 is a novel target of Ca2+-dependent CIPK–CBL complexes which tunes the pump activity affecting a stimulus-induced cytosolic Ca2+ transient in planta.

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 Ca2+-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-1M-I116) 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 (KD ∼10-fold higher than wild-type ACA8) and S99D (KD 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-1M-I116 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.

Ca2+ Pumps and Ca2+ Antiporters in Plant Development

Calcium (Ca2+) efflux transporters remove Ca2+ from the cytosol of the cell either by transporting it out of the cell across the plasma membrane or into internal organelles. These transporters, which include Ca2+-ATPases and Ca2+/H+ antiporters, have a critical role in preventing Ca2+ toxicity, maintaining cytosolic Ca2+ at a low resting level, and transferring Ca2+ to specific cellular locations where it is required. Many genes encoding plant Ca2+-ATPases and Ca2+/H+ antiporters have now been identified and characterised to elucidate their biochemical and genetic features. Furthermore, the use of gene knockouts has begun to provide evidence for an involvement of these Ca2+ transporters in Ca2+-signaling networks and in various aspects of plant development.

Plant and animal type 2B Ca2+-ATPases: evidence for a common auto-inhibitory mechanism.

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.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca 2+ ) sensor that interacts with the plasma membrane Ca 2+ -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.