A Ca 2ϩ -ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca 2ϩ -ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca 2ϩ -transporting P-type Ca 2ϩ -ATPases. The identity of At-ACA8p as the PM Ca 2ϩ -ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca 2ϩ -ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca 2ϩ -ATPase to be cloned and is clearly distinct from animal PM Ca 2ϩ -ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembranelocalized type IIB Ca 2ϩ -ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca 2ϩ -ATPases.

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.

Arabidopsis thalianaglutamate 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 inArabidopsisseedling 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 forl-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.

In plant Ca2؉ 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. Ca2ϩ acts as a secondary messenger in eukaryotic cells. One of the key proteins that mediate Ca 2ϩ signals is calmodulin (CaM) 2 a small, ubiquitous, and highly conserved protein found in all eukaryotes (1, 2). Upon Ca 2ϩ binding, CaM changes conformation, which enables CaM to bind to and regulate a wide array of target enzymes. The interaction between CaM and the CaM-binding domain (CaMBD) of the target enzyme is mainly hydrophobic in which two bulky hydrophobic residues in the CaMBD, the so-called anchor points, are especially important to anchor the protein to CaM. In addition, the complex is stabilized by electrostatic interactions between negatively charged glutamates in CaM and basic residues in the CaMBD (3-5). No consensus CaMBD exists for proteins that are targets of CaM, but CaMBDs typically have a hydrophobic and basic nature consisting of 15-30 amino acid residues that have a tendency to form an ␣-helix. Based on the position of the two hydrophobic anchor points, the majority of Ca 2ϩ -dependent CaMBDs is divided into three classes, namely 1-10, 1-14, and 1-16 (6, 7). Many CaM-regulated enzymes are autoinhibited with autoinhibition released by CaM. The autoinhibitory domain is often located either adjacent to or overlapping with the CaMBD (5, 8).The P 2B Ca 2ϩ -ATPases including PMCAs (plasma membrane Ca 2ϩ -ATPases) from animals and ACAs (autoinhibited Ca 2ϩ -ATPases) from plants are autoinhibitory proteins regulated by CaM. The regulatory region consisting of a CaMBD and an autoinhibitory domain is located in the C terminus in animals and N terminus in plants (9, 10). These two domains are likely to be at least partly overlapping in both plant and mammalian Ca 2ϩ -ATPases, as has been shown for the human PMCA4b (isoform 4, splice variant b) (11), Arabidopsis ACA2 (i...

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-calmodulinbinding 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. Role of cAMP in plant immune response 597Generation of Arabidopsis cAS-mCherry plants expressing the NES-YC3.6 probeThe Arabidopsis Col-0 cAS-transgenic lines were crossed with the Col-0 pUBQ10-NES-YC3.6 line reported in Krebs et al. (2012). Seeds from cross-pollinated flowers were surface sterilized by vapour-

A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the (794)HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.

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.

Edited by Joseph Jez 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 EFhands, 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 lowaffinity Ca 2؉-specific sites. Binding of Ca 2؉ induced an increase in the ␣-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. Calcium (Ca 2ϩ) is a crucial second messenger in plants, where it couples the perception of endogenous and environmental signals to plant responses (1-4). Ca 2ϩ signals are transmitted by stimulus-specific cytosolic Ca 2ϩ elevations that result from the concerted action of both Ca 2ϩ influx (channels) and Ca 2ϩ efflux (pumps and carriers) systems, which temporally shape and spatially define Ca 2ϩ dynamics, commonly This study was supported in part by Grant FUR2014 from the University of Verona (to A. A. and P. D.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Figs.