Identification of the Arabidopsis calmodulin-dependent NAD+ kinase that sustains the elicitor-induced oxidative burst

International audience17 NADP(H) is an essential cofactor of multiple metabolic processes in all living organisms. In plants, 18 NADP(H) is required as the substrate of Ca 2+-dependent NADPH oxidases which catalyze a reactive 19 oxygen species burst in response to various stimuli. While NADP + production in plants has long been 20 known to involve a Calmodulin and Calcium (CaM)/Ca 2+-dependent NAD + kinase, the nature of the 21 enzyme catalyzing this activity has remained enigmatic, as well as its role in plant physiology. Here, 22 thanks to a combination of proteomics, biochemistry, molecular biology and in vivo studies, we have 23 identified an Arabidopsis protein that catalyzes NADP + production exclusively in the presence of 24 CaM/Ca 2+. This new enzyme (NADKc) has a CaM-binding peptide located in its N-terminal region and 25 displays peculiar biochemical properties as well as different domain organization compared to known 26 plant NAD + kinases. In response to a pathogen elicitor, activity of NADKc, which is associated with the 27 mitochondrial periphery, contributes to an increase in the cellular NADP + concentration and to the 28 amplification of the elicitor-induced oxidative burst. Based on a phylogenetic analysis and enzymatic 29 assays, we propose that the CaM/Ca 2+-dependent NAD + kinase activity found in photosynthetic 3

La régulation des protéines plastidiales par la calmoduline

Calmodulin (CaM) is an important modulator of cell responses of eukaryotes. This protein is composed of four calcium (Ca2+)-binding sites and a flexible central helix. CaM can interact with other proteins in a Ca2+-dependent way. This leads to a wide variety of effects, such as activation/inhibition of enzymes, opening of membrane channels and regulation of protein trafficking. The identification of high-affinity CaM targets requires techniques allowing the study of the CaM-binding parameters of a large number of protein, and in several conditions mimicking the cell environment (e.g. presence of ligands or other proteins). The first objective of this PhD was to develop flexible and quantitative assays of CaM-partners interactions based on measurements of fluorescence anisotropy. these tests were used to perform a quantitative characterization of the interaction between CaM and two previously identified targets located in Arabidopsis chloroplast (NADK2 and Tic32). We then performed a high-throughput analysis (CaM-affinity chromatography coupled with mass spectrometry) in order to detect new potential plastidial CaM targets. We validated our approach with several biochemical techniques. We finally focused our attention on the ceQORH protein, whose high CaM affinity was confirmed by several tests. Our results confirm the Ca2+-dependent CaM affinity of NADK2, Tic32 and ceQORH and provide new elements for understanding the effects of these interactions. In addition, in order to verify the presence of CaMs or CaM-like proteins in the chloroplast, we used a biochemical and proteomic approach. We also studied the intracellular localization of some putative plastidial CMLs tagged with GFP in Arabidopsis protoplasts. For the moment, these approaches did not allow identifying such proteins in the chloroplast.La calmoduline (CaM) est une protéine modulatrice de la réponse cellulaire chez les eucaryotes composée de quatre domaines de liaison au calcium et d'une hélice centrale flexible. Elle peut interagir avec d'autres protéines en présence de calcium, entraînant l'activation et l'inhibition d'enzymes, l'ouverture de canaux membranaires et modulant le trafic intracellulaire. L'identification de protéines parternaires de la CaM requière la mise au point de techniques permettant de mesurer les paramètres de la liaison pour un grand nombre de protéines dans des conditions variables mimant l'environnement cellulaire (par exemple en présence de ligands ou d'autres protéines). Le premier objectif de cette thèse a été de développer une technique de mesure des interactions CaM-parternaire reposant sur des mesures d'anisotropie de fluorescence. Les tests ont été ensuite utilisés pour caractériser de manière quantitative l'interaction préalablement mise en évidence de deux protéines chloroplastiques (NADK2 et Tic32) avec la CaM. Afin d'identifier d'autres cibles chloroplastiques de la CaM nous avons alors effectué une analyse à haut-débit en couplant une purification par affinité à des analyses protéomiques. La validation des interactions a été réalisée grâce à l'utilisation de méthodes biochimiques complémentaires. Nous avons ensuite focalisé notre attention sur la protéine ceQORH dont la très forte affinité pour la CaM a pu être confirmée. Nos résultats fournissent par ailleurs de nouveaux éléments pour la compréhension de ces interactions. Afin de vérifier la présence de CaM ou de CaM-like (CML) dans le chloroplaste nous avons utilisé une approche biochimique et protéomique. Nous avons d'autre part étudié la localisation de CMLs potentiellement chloroplastiques fusionnées à la GFP dans des protoplastes d'Arabidopsis. A ce jour ces deux approches ne nous ont pas permis d'identifier ce type de protéines dans le chloroplaste

The regulation of plastidial proteins by calmodulins

La calmoduline (CaM) est une protéine modulatrice de la réponse cellulaire chez les eucaryotes composée de quatre domaines de liaison au calcium et d'une hélice centrale flexible. Elle peut interagir avec d'autres protéines en présence de calcium, entraînant l'activation et l'inhibition d'enzymes, l'ouverture de canaux membranaires et modulant le trafic intracellulaire. L'identification de protéines parternaires de la CaM requière la mise au point de techniques permettant de mesurer les paramètres de la liaison pour un grand nombre de protéines dans des conditions variables mimant l'environnement cellulaire (par exemple en présence de ligands ou d'autres protéines). Le premier objectif de cette thèse a été de développer une technique de mesure des interactions CaM-parternaire reposant sur des mesures d'anisotropie de fluorescence. Les tests ont été ensuite utilisés pour caractériser de manière quantitative l'interaction préalablement mise en évidence de deux protéines chloroplastiques (NADK2 et Tic32) avec la CaM. Afin d'identifier d'autres cibles chloroplastiques de la CaM nous avons alors effectué une analyse à haut-débit en couplant une purification par affinité à des analyses protéomiques. La validation des interactions a été réalisée grâce à l'utilisation de méthodes biochimiques complémentaires. Nous avons ensuite focalisé notre attention sur la protéine ceQORH dont la très forte affinité pour la CaM a pu être confirmée. Nos résultats fournissent par ailleurs de nouveaux éléments pour la compréhension de ces interactions. Afin de vérifier la présence de CaM ou de CaM-like (CML) dans le chloroplaste nous avons utilisé une approche biochimique et protéomique. Nous avons d'autre part étudié la localisation de CMLs potentiellement chloroplastiques fusionnées à la GFP dans des protoplastes d'Arabidopsis. A ce jour ces deux approches ne nous ont pas permis d'identifier ce type de protéines dans le chloroplaste.Calmodulin (CaM) is an important modulator of cell responses of eukaryotes. This protein is composed of four calcium (Ca2+)-binding sites and a flexible central helix. CaM can interact with other proteins in a Ca2+-dependent way. This leads to a wide variety of effects, such as activation/inhibition of enzymes, opening of membrane channels and regulation of protein trafficking. The identification of high-affinity CaM targets requires techniques allowing the study of the CaM-binding parameters of a large number of protein, and in several conditions mimicking the cell environment (e.g. presence of ligands or other proteins). The first objective of this PhD was to develop flexible and quantitative assays of CaM-partners interactions based on measurements of fluorescence anisotropy. these tests were used to perform a quantitative characterization of the interaction between CaM and two previously identified targets located in Arabidopsis chloroplast (NADK2 and Tic32). We then performed a high-throughput analysis (CaM-affinity chromatography coupled with mass spectrometry) in order to detect new potential plastidial CaM targets. We validated our approach with several biochemical techniques. We finally focused our attention on the ceQORH protein, whose high CaM affinity was confirmed by several tests. Our results confirm the Ca2+-dependent CaM affinity of NADK2, Tic32 and ceQORH and provide new elements for understanding the effects of these interactions. In addition, in order to verify the presence of CaMs or CaM-like proteins in the chloroplast, we used a biochemical and proteomic approach. We also studied the intracellular localization of some putative plastidial CMLs tagged with GFP in Arabidopsis protoplasts. For the moment, these approaches did not allow identifying such proteins in the chloroplast

The Pseudoenzyme PDX1.2 Sustains Vitamin B₆ Biosynthesis as a Function of Heat Stress

Plants sense temperature changes and respond by altering growth and metabolic activity to acclimate to the altered environmental conditions. The B vitamins give rise to vital coenzymes that are indispensable for growth and development but their inherent reactive nature renders them prone to destruction especially under stress conditions. Therefore, plant survival strategies would be expected to include mechanisms to sustain B vitamin supply under demanding circumstances. Here, using the example of vitamin B6, we investigate the regulation of biosynthesis across eudicot and monocot species under heat stress. Most eudicots carry a pseudoenzyme PDX1.2 that is a noncatalytic homolog of the PDX1 subunit of the vitamin B6biosynthesis protein machinery, PYRIDOXINE BIOSYNTHESIS PROTEIN1. Using Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) as models, we show thatPDX12is transcriptionally regulated by the HSFA1 transcription factor family. Monocots only carry catalyticPDX1homologs that do not respond to heat stress as demonstrated for rice (Oryza sativa) and maize (Zea mays), suggesting fundamental differences in the regulation of vitamin B6biosynthesis across the two lineages. Investigation of the molecular mechanism ofPDX12transcription reveals two alternative transcriptional start sites, one of which is exclusive to heat stress. Further data suggest that PDX1.2 leads to stabilization of the catalytic PDX1s under heat stress conditions, which would serve to maintain vitamin B6homeostasis in times of need in eudicots that carry this gene. Our analyses indicate an important abiotic stress tolerance strategy in several eudicots, which has not been evolutionarily adapted (or is not required) by monocots such as grasses

Adoção nacional e internacional: significados, motivações e processos de habilitação

Through a multiple case study - a domestic and an international adoption - the present study investigated meanings, motivations, and qualification processes in adoption cases. The methodology used was the Ecological Engagement, in which interviews, observations, and visits to the families were conducted. Adoption was perceived as an action directed to the child's welfare in both cases. In the domestic case, the main motivation for adoption related to the affective bond formed by children and adoptees in their prior contact, whereas in the international case it related to problems in pregnancy and having biological children. The way adoptees signify the adoption process as well as values and beliefs present in society influence how adoption occurs.A partir de um estudo de casos múltiplos - uma adoção nacional e outra internacional - este estudo investigou as significações, motivações e o processo de habilitação em casos de adoção. Utilizou-se a metodologia da Inserção Ecológica, na qual entrevistas, observações e visitas às famílias foram conduzidas. A adoção foi percebida como uma ação voltada para o bem da criança em ambos os casos. No caso nacional, a motivação para adoção se deu pelo vínculo afetivo existente, criado através do contato anterior com as crianças, e no internacional por dificuldade para manter uma gravidez e ter filhos biológicos. A forma como os adotantes significam processo de adoção, bem como os valores e ideias presentes na sociedade influenciam em como se experiencia a adoção.Através de un estudio de caso múltiple - una adopción nacional y otra internacional - este estudio investigó los significados, motivación y habilitación para adopción. Se utilizó la metodología de Inserción Ecológica, en el que entrevistas, observaciones y visitas a familia se llevaron a cabo. La adopción fue percibida como una acción dirigida hacia el bien del niño en ambos casos. En el caso nacional, la motivación para la adopción estaba relacionada con el vínculo afectivo creado por un contacto previo con los niños y en el caso internacional a dificultades para mantener un embarazo y tener hijos biológicos. La forma en que los adoptantes dan significado al proceso de adopción, así como los valores y las ideas en la sociedad influyen en la experiencia de la adopción

Adoçao nacional e internacional: processos proximais no período de convivência

This study investigated the period of cohabitation of children/adolescents and their adopters in domestic and international adoptions� processes, through a multiple case study. The methodology used was the Ecological Engagement, by monitoring the families for about four month, interviews, observations, visits to the shelter�s institutions and a meeting in Italy three months after the departure of the children. Data was organized into themes based on Bioecological theory (PPCT model). In the domestic adoption the proximal processes were facilitated by the prior knowledge of the child/adolescent, and in the international adoption by the adopters� perception of children�s engagement in the adoption. The dysfunctional processes in the domestic case were related to the youngest child behavior�s change, while in the international to the fact of being in an unfamiliar environment. It can be concluded that the adoptions� processes are complex, and the period of cohabitation is critical to the family adaptation, so that it should be performed with greater availability of psychosocial support in its various stages, especially in an initial moment of adaptation and changes and considering the particularities of each case.Este estudio investigó el período de convivencia de niños/adolescentes y sus adoptantes en proceso de adopción nacional e internacional, a través de un estudio de casos múltiples. Se utilizó la metodología de la Inserción Ecológica con acompañamiento de las familias durante unos cuatro meses, entrevistas, observaciones, visitas a las instituciones de acogimiento y una visita en Italia tres meses después de la salida de los niños. Los datos fueron organizados en temas basados en la Teoría bioecológica (modelo PPCT). En la adopción nacional los procesos proximales fueron facilitados por el conocimiento anterior de niño/adolescente, y en la adopción internacional por la percepción del compromiso de los niños en la adopción. Los procesos disfuncionales en el caso nacional estaban relacionados con el cambio de comportamiento de la niña más pequeña, mientras en el internacional al hecho de estar en un ambiente desconocido. Se puede concluir que los procesos de adopción son complejos, y el período de convivencia fundamental para la adaptación de la familia, así se debe realizar con una mayor disponibilidad de apoyo psicosocial en sus diversas etapas, principalmente en un momento inicial de adaptación y cambio, tiendo en cuenta las particularidades de cada caso

Complementary biochemical approaches applied to the identification of plastidial calmodulin-binding proteins.

International audienceCa(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM