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.

In developed countries, obesity is the most frequent nutritional disorder, and overweight and obesity prevalences have increased whilst physical activity and breakfast consumption have declined. There is growing scientific interest in the possible role of breakfast in weight control and in factors such as appetite control, dietary quality and reduced risk for chronic diseases. The current article reviews the literature and discusses how the breakfast 'environment' and the composition of breakfast meals might be improved, particularly in children, so as to maintain the breakfast eating habit throughout life. Recommendations are proposed to encourage children to keep eating breakfast and the nutritional composition of the 'American breakfast' and two types of Mediterranean 'cereal breakfasts' are compared. We also propose a new breakfast type for children and adolescents that is based on chocolate hazelnut spread within a mixed breakfast type in order to reinforce positive experiences.

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

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.

In planta, very limited information is available about how the endoplasmic reticulum (ER)

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.

SignificancePlant roots grow toward water, a phenomenon termed hydrotropism. The nature of the root signal that governs hydrotropism is elusive and remains to be elucidated. Here, we show that, in response to water potential differences across the root tip, a slow, asymmetric long-distance Ca2+ signal is transmitted via the phloem to the elongation zone, where it is asymmetrically distributed across the root to promote root curvature. Furthermore, we demonstrate that the hydrotropism-associated protein MIZ1 plays a role in generating the Ca2+ signal by its direct binding to and inhibition of ECA1, an endoplasmic reticulum Ca2+ pump, which resembles the animal sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). This study elucidates the mechanism underlying systemic Ca2+ signaling in plant roots, which is essential for water tracking.