Functional Interchangeability of Nucleotide Sugar Transporters URGT1 and URGT2 Reveals That urgt1 and urgt2 Cell Wall Chemotypes Depend on Their Spatio-Temporal Expression

[EN] Nucleotide sugar transporters (NSTs) are Golgi-localized proteins that play a role in polysaccharide biosynthesis by transporting substrates (nucleotide sugars) from the cytosol into the Golgi apparatus. In Arabidopsis, there is an NST subfamily of six members, called URGTs, which transport UDP-rhamnose and UDP-galactose in vitro. URGTs are very similar in protein sequences, and among them, URGT1 and URGT2 are highly conserved in protein sequence and also showed very similar kinetic parameters toward UDP-rhamnose and UDP-galactose in vitro. Despite the similarity in sequence and in vitro function, mutants in urgt1 led to a specific reduction in galactose in rosette leaves. In contrast, mutants in urgt2 showed a decrease in rhamnose content in soluble mucilage from seeds. Given these specific and quite different chemotypes, we wonder whether the differences in gene expression could explain the observed differences between the mutants. Toward that end, we analyzed whether URGT2 could rescue the urgt1 phenotype and vice versa by performing a promoter swapping experiment. We analyzed whether the expression of the URGT2 coding sequence, controlled by the URGT1 promoter, could rescue the urgt1 rosette phenotype. A similar strategy was used to determine whether URGT1 could rescue the urgt2 mucilage phenotype. Expression analysis of the swapped genes, using qRT-PCR, was similar to the native URGT1 and URGT2 genes in wild-type plants. To monitor the protein expression of the swapped genes, both URGTs were tagged with green fluorescent protein (GFP). Confocal microscopy analyses of the swapped lines containing URGT2-GFP showed fluorescence in motile dot-like structures in rosette leaves. Swapped lines containing URGT1-GFP showed fluorescence in dot-like structures in the seed coat. Finally, the expression of URGT2 in urgt1 mutants rescued galactose reduction in rosette leaves. In the same manner, the expression of URGT1 in urgt2 mutants recovered the content of rhamnose in soluble mucilage. Hence, our results showed that their expression in different organs modulates the role in vivo of URGT1 and URGT2. Likely, this is due to their presence in different cellular contexts, where other proteins, acting in partnership, may drive their functions toward different pathways.SIThis work was supported by FONDECYT 11160787 and 1204467 (SS-A), 1151335 (AO), and 3170796 (AL-G), FONDAP- Centro de Regulación del Genoma 15090007 (AO), ECOSud-CONICYT C14B02 (AO), and PAI 79170136 (SS-A)

GoSAMTs are required for pectin methyl-esterification and mucilage release in seed coat epidermal cells.

Peer reviewed: TrueINTRODUCTION: GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. METHODS: To determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. RESULTS: We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of "egg-box" structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. DISCUSSION: The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants

Transport of UDP-Rhamnose by URGT2, URGT4 and URGT6 modulates Rhamnogalacturonan-I length

International audienceRhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of soluble mucilage polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in RG-I elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage. Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains

Las habilidades para la vida en la clase de educación física

Tesis (Profesor de Educación Física para la Enseñanza Básica, Licenciado en Educación)Esta investigación profundizará sobre el tema de las habilidades para la vida, que son aquellas aptitudes necesarias para tener un comportamiento adecuado y positivo que permite enfrentar eficazmente los retos de la vida diaria. Así descritas son elementos constitutivos fundamentales del desarrollo de habilidades personales para la promoción de la salud señaladas como un área de acción clave en la carta de Ottawa en 1986. El desarrollo de habilidades para la vida ha formado parte de los programas de adolescentes alrededor del mundo, ya sea como elemento de desarrollo de los jóvenes, formación de valores, prevención de embarazos precoces, planificación de la vida, aprendizaje social y emocional, iniciativas para la prevención de abuso de drogas. Organismos internacionales como la UNICEF, la Organización Mundial de la Salud (OMS) y la Organización Panamericana de la Salud (OPS), han apoyado iniciativas que fomentan el desarrollo de este tipo de programas desde hace ya varios años. La estrategia de Habilidades para la Vida constituye una importante metodología para promover la salud, la educación y la formación valórica y social de los adolescentes, por esta razón queremos saber ¿Qué ocurre con los profesores de educación física a la hora de enseñar y aplicar las habilidades para la vida durante el desarrollo de sus clases? La Educación Física constituye una asignatura que integra acciones, principalmente, de carácter motriz. Su organización y planificación en los diferentes niveles de enseñanza está sujeta a las características de los alumnos hacia los cuales está dirigida, siempre tomando en cuenta la formación personal e integral de los alumnos. En este sentido es preciso y lógico señalar que su expresión como actividad propia de un proceso pedagógico depende en gran medida de la metodología de los docentes y de los requerimientos que demanda la sociedad para la formación de los alumnos. De esta manera a través de una pauta de observación buscamos indagar respecto a la implementación de las habilidades para la vida en la clase de educación física

Relationship between mechanical and metabolic dyssynchrony with left bundle branch block: Evaluation by 18-fluorodeoxyglucose positron emission tomography in patients with non-ischemic heart failure

Background: Ventricular dyssynchrony is a common finding in patients with heart failure (HF), especially in the presence of conduction delays. The loss of ventricular synchrony leads to progressive impairment of contractile function, which may be explained in part by segmental abnormalities of myocardial metabolism. However, the association of these metabolic disarrangements with parameters of ventricular dyssynchrony and electrocardiography (ECG) findings has not yet been studied. Methods: Our aim was to determine the correlation between the presence of left bundle branch block (LBBB) with left ventricular (LV) mechanical synchrony assessed by multiple-gated acquisition scan (MUGA) and with patterns of 18-fluorodeoxyglucose (18FDG) uptake in patients with non-ischemic heart failure. Twenty-two patients with non-ischemic cardiomyopathy, LV ejection fraction (LVEF) ≤45% and New York Heart Association (NYHA) Functional Class II or III symptoms under standard medical therapy were include

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective