Developing technologies to assess vascular ageing: a roadmap from VascAgeNet.

Vascular ageing is the deterioration of arterial structure and function which occurs naturally with age, and which can be accelerated with disease. Measurements of vascular ageing are emerging as markers of cardiovascular risk, with potential applications in disease diagnosis and prognosis, and for guiding treatments. However, vascular ageing is not yet routinely assessed in clinical practice. A key step towards this is the development of technologies to assess vascular ageing. In this Roadmap, experts discuss several aspects of this process, including: measurement technologies; the development pipeline; clinical applications; and future research directions. The Roadmap summarises the state of the art, outlines the major challenges to overcome, and identifies potential future research directions to address these challenges.

School-based soccer practice is an effective strategy to improve cardiovascular and metabolic risk factors in overweight children.

We examined the effects of a 6-month school-based soccer programme on cardiovascular (CV) and metabolic risk factors in overweight children. Methods: 40 boys [8-12 years; body mass index (BMI) >2 standard deviations of WHO reference values] participated in complementary school-based physical education classes (two sessions per week, 45-90 min each). The participants were divided into a soccer group (SG; n = 20) and a control group (CG; n = 20). The SG intervention involved 3 extra-curricular school-based soccer sessions per week, 60-90 min each. The intervention lasted for 6-months. All measurements were taken at baseline and after 6-months. From baseline to 6-months, the SG significantly improved (p < .05) BMI z-score, waist circumference, waist-to-height ratio, percentage of fat mass, percentage of fat-free mass, diastolic blood pressure, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol, but no such improvements were observed for the CG. After the intervention, the prevalence of soccer participants with normal waist-to-height ratio (30 vs. 5%; p = .037), systolic blood pressure (90 vs. 55%; p = .039), total cholesterol (80 vs. 65%; p = .035) and LDL-C (90 vs. 75%; p = .012) were significantly higher than at baseline. The findings suggest that a 6-month school-based soccer intervention program represents an effective strategy to reduce CV and metabolic risk factors in overweight children prepared to take part in a soccer program.

Characterization of plant glutamine synthetase S-nitrosation.

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

Effects of 6-month soccer and traditional physical activity programmes on body composition, cardiometabolic risk factors, inflammatory, oxidative stress markers and cardiorespiratory fitness in obese boys.

Physical activity is important in obesity prevention, but the effectiveness of different physical activity modalities remains to be determined among children. The main purpose of this study was to compare the effects of a 6-month soccer programme and a traditional physical activity programme on changes in body composition, cardiometabolic risk factors, inflammatory and oxidative markers, cardiorespiratory fitness and perceived psychological status in obese boys. Eighty-eight boys (8-12 years; BMI > +2 standard deviations of WHO reference values) participated in one of three groups: soccer, traditional activity and control. Soccer and traditional activity programmes involved 3 sessions per week for 60-90 min at an average intensity of 70-80% of maximal heart rate. Control group participated in activities of normal daily living. All boys participated in school physical education, two sessions per week of 45-90-min. Measurements were taken at baseline and after 6 months, and included body size and composition, cardiometabolic risk factors, inflammatory and oxidative markers, cardiorespiratory fitness and perceived psychological status. Physical activity and dietary intake were assessed before and immediately following the intervention. The three groups had similar characteristics at baseline. After 6 months, both intervention groups had significantly lower relative fatness (% fat), waist circumference and total cholesterol, and higher cardiorespiratory fitness, self-esteem, perceived physical competence and attraction to physical activity compared with control group. In conclusion, physical activity interventions over 6 months positively influenced several indicators of health status among obese boys. The results also suggested that soccer has the potential as an effective tool for the prevention and reduction of childhood obesity and associated consequences.

Possible role of glutamine synthetase of the prokaryotic type (GSI-like) in nitrogen signaling in Medicago truncatula.

Genes containing domains related to glutamine synthetase of the prokaryotic type (GSI-like) are widespread in higher plants, but their function is currently unknown. To gain insights into the possible role of GSI-like proteins, we characterized the GSI-like gene family of Medicago truncatula and investigated the functionality of the encoded proteins. M. truncatula contains two-expressed GSI-like genes, MtGSIa and MtGSIb, encoding polypeptides of 454 and 453 amino acids, respectively. Heterologous complementation assays of a bacterial glnA mutant indicate that the proteins are not catalytically functional for glutamine synthesis. Gene expression was investigated by qRT-PCR and western blot analysis in different organs of the plant and under different nitrogen (N) regimes, revealing that both genes are preferentially expressed in roots and root nodules, and that their expression is influenced by the N-status of the plant. Analysis of transgenic plants expressing MtGSI-like-promoter-gusA fusion, indicate that the two genes are strongly expressed in the root pericycle, and interestingly, the expression is enhanced at the sites of nodule emergence being particularly strong in specific cells located in front of the protoxylem poles. Taken together, the results presented here support a role of GSI-like proteins in N sensing and/or signaling, probably operating at the interface between perception of the N-status and the developmental processes underlying both root nodule and lateral root formation. This study indicates that GSI-like genes may represent a novel class of molecular players of the N-mediated signaling events.

Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme.

Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

Percepciones de los estudiantes de psicología sobre las relaciones interpersonales en el ámbito universitario: un estudio exploratorio / Percepções de alunos de psicologia sobre as relações interpessoais no ambiente universitário: um estudo exploratório / Perceptions of psychology students of interpersonal relationships in the college environment: an exploratory study

Esta propuesta tiene como objetivo identificar las percepciones de graduados a respecto de las relaciones sociales establecidas con la comunidad académica. Fue utilizada la técnica de Grupo Focal y Análisis de Contenido para cosecha y análisis de datos. Fueron listados dos niveles de dificultad relativos a las situaciones vivenciadas por los alumnos. Resalté la integración de los alumnos, la relación solícita entre los alumnos y profesores, funcionarios y gestores como fáciles; las subcategorías de la intolerancia y de la falta de tiempo para el relacionamiento entre los alumnos, la falta de compromiso docente, la arrogancia y descaso de los funcionarios y la valorización de la pos en detrimento de la graduación como difíciles. Se sugiere el desarrollo de programas en habilidades sociales con participantes del medio académico destacándose la escasez de trabajos con segmentos técnicos administrativos.

Objetivou-se identificar as percepções de graduandos a respeito das relações sociais estabelecidas com a comunidade acadêmica. Utilizou-se a técnica de Grupo Focal e Análise de Conteúdo para coleta e análise dos dados. Foram elencados dois níveis de dificuldade relativos às situações vivenciadas pelos alunos. Destacam-se a integração de alunos, a relação solícita entre alunos e professores, funcionários e gestores como fáceis; as subcategorias da intolerância e da falta de tempo para o relacionamento entre os alunos, a falta de compromisso docente, a arrogância e descaso dos funcionários e a valorização da pós em detrimento da graduação como difíceis. Sugere-se o desenvolvimento de programas em habilidades sociais com participantes do meio acadêmico destacando-se a escassez de trabalhos com segmentos técnicos administrativos.

This article aims to identify the perceptions of undergraduates about the social relationships established in the academic community. Focus Group and Content Analysis were used for data collection and analysis. Two difficulty levels related to the situations experienced by students were listed. It is noteworthy that the subcategories of integration among the students, the good relationship between students and professors, staff and managers were considered easy situations; and subcategories of intolerance and lack of time for the relationship between the students, the lack of commitment of professors, the arrogance and indifference of the staff, and the appreciation of post-graduation over graduation were classified as difficult situations. We suggest the development of training programs in social skills with participants from the academic community, a rich environment of social interactions, highlighting in particular the shortage of work in this regard with the segment of administrative staff.

Inteligência, autoeficácia e habilidades sociais em estudantes universitários / Intelligence, self-efficacy, and social skills in college students / Inteligencia, autoeficacia y habilidades sociales en estudiantes universitarios

Considerando a importância das habilidades comportamentais e cognitivas no desenvolvimento acadêmico e social dos estudantes, esta pesquisa tem por objetivo verificar a existência de relações entre autoeficácia, inteligência e habilidades sociais de 162 universitários e identificar as diferenças de gênero e tipo de instituição. Foram utilizados o Inventário de Habilidades Sociais (IHS), o Teste de Raciocínio Abstrato da Bateria de Provas de Raciocínio (BPR-5) e a Escala de Autoeficácia Acadêmica Percebida (EAEAP). A pesquisa indicou correlações entre habilidades sociais e autoeficácia e as habilidades sociais se mostraram dissociadas da inteligência fluida. O estudo apontou que gênero e tipo de instituição de ensino estão relacionados ao uso de diferentes habilidades cognitivas e/ou comportamentais nas vivências acadêmicas dos estudantes.

Considering the importance of behavioral and cognitive skills in academic and social development of students, this research aims to verify whether there is any relationship between self-efficacy, intelligence and social skills of 162 university students, as well as identify differences by gender and type of institution. This research used the Social Skills Inventory (IHS), the BPR-5's Abstract Reasoning Test, and the Perceived Academic Self-Efficacy Scale (EAEAP). The research showed correlations between social skills and self-efficacy, while social skills proved decoupled from fluid intelligence. The study also found that students use different cognitive or behavioral skills to adapt their academic experiences according to their gender and type of institution.

Considerando la importancia de las habilidades comportamentales y cognitivas en el desarrollo académico y social de los estudiantes, esta investigación tiene por objetivo verificar la existencia de relaciones entre autoeficacia, inteligencia y habilidades sociales de 162 universitarios e identificar las diferencias de género y tipo de institución. Fueron utilizados el Inventario de Habilidades Sociales (IHS), el Test de Raciocinio Abstracto de la Batería de Pruebas de Raciocinio (BPR-5) y la Escala de Autoeficacia Académica Percibida (EAEAP). La investigación indicó correlaciones entre habilidades sociales y autoeficacia, y las habilidades sociales se mostraron disociadas de la inteligencia fluida. El estudio indicó qué género y tipo de institución de enseñanza están relacionados al uso de diferentes habilidades cognitivas y/o comportamentales en las vivencias académicas de los estudiantes.

Inteligência, autoeficácia e habilidades sociais em estudantes universitários / Intelligence, self-efficacy, and social skills in college students / Inteligencia, autoeficacia y habilidades sociales en estudiantes universitarios

Considerando a importância das habilidades comportamentais e cognitivas no desenvolvimento acadêmico e social dos estudantes, esta pesquisa tem por objetivo verificar a existência de relações entre autoeficácia, inteligência e habilidades sociais de 162 universitários e identificar as diferenças de gênero e tipo de instituição. Foram utilizados o Inventário de Habilidades Sociais (IHS), o Teste de Raciocínio Abstrato da Bateria de Provas de Raciocínio (BPR-5) e a Escala de Autoeficácia Acadêmica Percebida (EAEAP). A pesquisa indicou correlações entre habilidades sociais e autoeficácia e as habilidades sociais se mostraram dissociadas da inteligência fluida. O estudo apontou que gênero e tipo de instituição de ensino estão relacionados ao uso de diferentes habilidades cognitivas e/ou comportamentais nas vivências acadêmicas dos estudantes.(AU)

Considering the importance of behavioral and cognitive skills in academic and social development of students, this research aims to verify whether there is any relationship between self-efficacy, intelligence and social skills of 162 university students, as well as identify differences by gender and type of institution. This research used the Social Skills Inventory (IHS), the BPR-5's Abstract Reasoning Test, and the Perceived Academic Self-Efficacy Scale (EAEAP). The research showed correlations between social skills and self-efficacy, while social skills proved decoupled from fluid intelligence. The study also found that students use different cognitive or behavioral skills to adapt their academic experiences according to their gender and type of institution.(AU)

Considerando la importancia de las habilidades comportamentales y cognitivas en el desarrollo académico y social de los estudiantes, esta investigación tiene por objetivo verificar la existencia de relaciones entre autoeficacia, inteligencia y habilidades sociales de 162 universitarios e identificar las diferencias de género y tipo de institución. Fueron utilizados el Inventario de Habilidades Sociales (IHS), el Test de Raciocinio Abstracto de la Batería de Pruebas de Raciocinio (BPR-5) y la Escala de Autoeficacia Académica Percibida (EAEAP). La investigación indicó correlaciones entre habilidades sociales y autoeficacia, y las habilidades sociales se mostraron disociadas de la inteligencia fluida. El estudio indicó qué género y tipo de institución de enseñanza están relacionados al uso de diferentes habilidades cognitivas y/o comportamentales en las vivencias académicas de los estudiantes.(AU)

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.

The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

Validation and factorial invariance of children's attraction to physical activity (CAPA) scale in Portugal.

The Children's Attraction to Physical Activity (CAPA) scale assesses interest in and attraction to the physical activity (PA) of children of elementary school age. The original (25 items) and shorter versions (15 items) of the scale were developed and validated with American children. The purpose of this study was to cross-validate the shorter version of the CAPA scale for use with Portuguese schoolchildren and to examine the invariance of the multidimensional factor structure of the scale in two samples. The sample comprised 683 children (7-10 years) from public primary schools. The sample was divided into calibration and cross-validation samples. The scale was translated into Portuguese and underwent forward translation, synthesis of the translation and backward translation and was then subjected to expert committee review, pretest and reliability assessment. Internal consistency for each of the five subscales within the a priori 5-factor structure of the CAPA scale was evaluated through Cronbach's alpha, followed by a series of confirmatory factor analyses (CFAs) for both the calibration and cross-validation samples. The maximum likelihood robust estimation method was used. The CFA demonstrated that a 5-factor structural model of the Portuguese translation of the CAPA scale was invariant. The construct analysed had the same basic meaning and structural and item differences within the two samples. The results indicated that the CAPA scale is appropriate for use with Portuguese schoolchildren. The availability of a valid and reliable scale should enhance opportunities for further understanding of children's involvement in PA.

Novel aspects of glutamine synthetase (GS) regulation revealed by a detailed expression analysis of the entire GS gene family of Medicago truncatula under different physiological conditions.

BACKGROUND: Glutamine Synthetase (GS, EC 6.3.1.2) is a central enzyme in nitrogen metabolism, and a key component of nitrogen use efficiency (NUE) and plant yield and thus it is extremely important to understand how it is regulated in plants. Medicago truncatula provides an excellent model system to study GS, as it contain a very simple GS gene family comprising only four expressed genes, MtGS1a and MtGS1b encoding cytosolic polypeptides, and MtGS2a and MtGS2b encoding plastid-located enzymes. To identify new regulatory mechanisms controlling GS activity, we performed a detailed expression analysis of the entire GS gene family of M. truncatula in the major organs of the plant, over a time course of nodule or seed development and during a diurnal cycle. RESULTS: Individual GS transcripts were quantified by qRT-PCR, and GS polypeptides and holoenzymes were evaluated by western blot and in-gel activity under native electrophoresis. These studies revealed that all four GS genes are differentially regulated in each organ of the plant, in a developmental manner, and identified new regulatory controls, which appear to be specific to certain metabolic contexts. Studies of the protein profiles showed that the GS polypeptides assemble into organ-specific protein complexes and suffer organ-specific post-translational modifications under defined physiological conditions. Our studies also reveal that GS expression and activity are modulated during a diurnal cycle. The biochemical properties of the four isoenzymes were determined and are discussed in relation to their function in the plant. CONCLUSIONS: This work provides a comprehensive overview of GS expression and regulation in the model legume M. truncatula, contributing to a better understanding of the specific function of individual isoenzymes and to the identification of novel organ-specific post-translational mechanisms of GS regulation. We demonstrate that the GS proteins are modified and/or integrated into protein-complexes that assemble into a specific composition in particular organs of the plant. Taken together, the results presented here open new avenues to explore the regulatory mechanisms controlling GS activity in plants, a subject of major importance due to the crucial importance of the enzyme for plant growth and productivity.

Psychosocial correlates of physical activity in school children aged 8-10 years.

BACKGROUND: Understanding correlates of physical activity (PA) among children in different populations may contribute to fostering active lifestyles. This study considered gender differences in relationships between biologic (body mass index, BMI), demographic (socioeconomic sport status, SES) and psychosocial correlates of PA and level of PA in Portuguese primary school children. METHODS: 683 children, aged 8-10 years, from 20 different elementary schools in northern Portugal were surveyed. Weight status was classified using International Obesity Task Force (IOTF) criteria for the BMI. Family SES was estimated from school records. PA level and psychosocial correlates (attraction to PA, perceived physical competence and parental socialization) were obtained with interview and standardized questionnaires, respectively. Sex-specific hierarchical multiple regression analyses (SPSS 18.0) were conducted and included two blocks of predictor variables (biologic and demographic, and psychosocial). RESULTS: Level of PA was significantly higher in boys than girls. Enjoyment of participation in vigorous PA was positively associated with level of PA. Perceived acceptance by peers in games and sports and parental encouragement were positively and significantly related to PA in girls. Perceived physical competence was positively and significantly related to PA in boys. Weight status and SES were not associated with PA. CONCLUSIONS: Boys and girls differed in perceived attractiveness of PA and perceived physical competence, both of which influenced level of PA. Differences in perceptions may be important aspects of motivation for PA in school children.

Gender, weight status and socioeconomic differences in psychosocial correlates of physical activity in schoolchildren.

OBJECTIVES: This study sought to assess differences in attraction to physical activity, perceived physical competence and parental socialization influences across gender, body mass index and socioeconomic status in Portuguese children. DESIGN: Cross-sectional study. METHODS: 683 children, aged 8-10 years, from elementary schools were participants. Attraction to physical activity, perceived physical competence, parental socialization influences and socioeconomic status were assessed via standardized questionnaires. The prevalence of overweight and obesity was calculated using body mass index, based on the international cut-off points. MANOVA and ANOVA models were conducted. RESULTS: Boys reported greater enjoyment of games and sports participation than did girls. Boys and normal-weight children perceived themselves as being more successful and physically competent than did girls and obese children. Normal-weight girls enjoyed participation in vigorous physical activity more than did overweight and obese girls. Obese children felt less accepted by their peers in games and sports than did normal-weight and overweight children. High and medium socioeconomic status children perceived physical activity participation as of greater importance than did low-socioeconomic status children. High-socioeconomic status girls reported greater liking of the exertional aspects of physical activity compared to low socioeconomic status girls. High socioeconomic status children were more likely to perceive their parents as positive role models and perceived that they had greater enjoyment of physical activity than did lower socioeconomic status children. CONCLUSIONS: These results suggest that physical activity promotion interventions should focus on girls, obese children and lower socioeconomic status children as these individuals tend to have lower levels of attraction to physical activity, lower perceived physical competence and less parent physical activity support, which puts them at greater risk of being physically inactive.

Evaluating the youth physical activity promotion model among Portuguese elementary schoolchildren.

BACKGROUND: The Youth Physical Activity Promotion (YPAP) model provides an integrated approach to understanding the predisposing, enabling, and reinforcing factors influencing physical activity (PA) behavior. The purpose of this study was to evaluate an adapted version of the YPAP model for explaining PA among Portuguese schoolchildren. METHODS: A random cross-sectional sample of 683 children (8-10 years of age) attending elementary public schools in the north of Portugal completed a detailed survey assessing attraction to PA, perceived physical competence, parental influences and leisure time PA. Structural equation modeling techniques were conducted (EQS6.1). RESULTS: Attraction to PA was directly associated with children's PA participation (ß = 0.271, P < .05). Perceived physical competence imposed an indirect effect on children's PA through children's attraction to PA (ß = 0.253, P < .05). Parental influence had an indirect effect on children's PA through perceived physical competence and attraction to PA (ß = 0.318 and 0.662, respectively, P < .05). Perceived physical competence and parental influence were not directly associated with children's PA (ß = 0.069 and 0.180, respectively, P > .05). CONCLUSIONS: The adapted version of YPAP model was useful in explaining PA participation in elementary Portuguese schoolchildren. Intervention programs intended to enhance attraction to PA, perceived physical competence and favorable parental influence should be developed to promote children's PA participation.

Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatula.

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Glutamine synthetase is a molecular target of nitric oxide in root nodules of Medicago truncatula and is regulated by tyrosine nitration.

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds.

BACKGROUND: Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. RESULTS: This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. CONCLUSIONS: This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.

Crystallization and preliminary crystallographic characterization of glutamine synthetase from Medicago truncatula.

The condensation of ammonium and glutamate into glutamine catalyzed by glutamine synthetase (GS) is a fundamental step in nitrogen metabolism in all kingdoms of life. In plants, this is preceded by the reduction of inorganic nitrogen to an ammonium ion and therefore effectively articulates nitrogen fixation and metabolism. Although the three-dimensional structure of the dodecameric bacterial GS was determined quite some time ago, the quaternary architecture of the plant enzyme has long been assumed to be octameric, mostly on the basis of low-resolution electron-microscopy studies. Recently, the crystallographic structure of a monocotyledonous plant GS was reported that revealed a homodecameric organization. In order to unambiguously establish the quaternary architecture of GS from dicotyledonous plants, GS1a from the model legume Medicago truncatula was overexpressed, purified and crystallized. The collection of synchrotron diffraction data to 2.35 A resolution allowed the determination of the three-dimensional structure of this enzyme by molecular replacement.

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula.

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.