Activation of plasma membrane H + -ATPases participates in dormancy alleviation in sunflower seeds

International audienceUsing various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3′ end processing, through the regulation of SA production, is a key component of plant immune responses

The Association between Intrauterine Inflammation and Spontaneous Vaginal Delivery at Term: A Cross-Sectional Study

BACKGROUND:Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor. METHODS/RESULTS:In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher (18 vs 4%, p = 0.02) and amniotic fluid concentration of IL-6 was higher (3.1 vs 0.37 ng/mL, p<0.001). Similar results were obtained for IL-8 (10.93 vs 0.96 ng/mL, p<0.001) and percentage of detectable TNF-alpha (50 vs 4%, p<0.001). CONCLUSIONS:This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines

Importance de l'homéostasie du NAD dans la productivité et la résistance aux stress chez Arabidospis thaliana

The harmonious development and functioning of plants depend on severalcofactors such asnicotinamide adenine dinucleotide. Besides its roles in redox recycling,the NAD is alsoinvolved in cellular signalling processes, which are central actors instress situations. TheNAD is thus one of the main determinants of plant energy homeostasis,and therefore of cropyield. Through a reverse genetics approach targeting the L-aspartateoxidase (AO) – the firstcommitted enzyme of NAD biosynthesis – plants with constitutive eitherincreased ordecreased levels of pyridine nucleotides have been obtained. A furtherstudy of these plantsenabled to show that the NAD (1) can improve the growth, development andproductivity ofplants, (2) coordinates photosynthetic, respiratory and nitrogenmetabolisms, and (3) actsupon biotic and abiotic stress resistance mechanisms. This research isbeing valuedindustrially.Le développement et le fonctionnement harmonieux des plantes dépendentde cofacteurscomme le nicotinamide adénine dinucléotide (NAD). Outre ses rôles dansle recyclage rédox,le NAD est aussi impliqué dans des processus de signalisationcellulaire, particulièrementsollicités en situation de stress. Cela fait du NAD l’un desdéterminants majeurs del’homéostasie énergétique des plantes et donc des rendements descultures. Par uneapproche de génétique inverse ciblant la L-aspartate oxydase (AO),première enzyme de lavoie de biosynthèse du NAD, nous avons obtenu des plantes aux teneursconstitutivementaugmentées et diminuées en nucléotides à pyridine. L’étude de cesplantes a permisd’établir que (1) le NAD stimule la croissance, le développement et laproductivité desplantes, (2) coordonne les métabolismes photosynthétique, respiratoireet azoté, et (3)intervient dans les mécanismes de résistance aux stress biotiques etabiotiques. Les travauxcités ci-dessus font l’objet d’une valorisation industrielle

Importance of NAD homeostasis in productivity and stress resistance in Arabidopsis thaliana

Le développement et le fonctionnement harmonieux des plantes dépendentde cofacteurscomme le nicotinamide adénine dinucléotide (NAD). Outre ses rôles dansle recyclage rédox,le NAD est aussi impliqué dans des processus de signalisationcellulaire, particulièrementsollicités en situation de stress. Cela fait du NAD l’un desdéterminants majeurs del’homéostasie énergétique des plantes et donc des rendements descultures. Par uneapproche de génétique inverse ciblant la L-aspartate oxydase (AO),première enzyme de lavoie de biosynthèse du NAD, nous avons obtenu des plantes aux teneursconstitutivementaugmentées et diminuées en nucléotides à pyridine. L’étude de cesplantes a permisd’établir que (1) le NAD stimule la croissance, le développement et laproductivité desplantes, (2) coordonne les métabolismes photosynthétique, respiratoireet azoté, et (3)intervient dans les mécanismes de résistance aux stress biotiques etabiotiques. Les travauxcités ci-dessus font l’objet d’une valorisation industrielle.The harmonious development and functioning of plants depend on severalcofactors such asnicotinamide adenine dinucleotide. Besides its roles in redox recycling,the NAD is alsoinvolved in cellular signalling processes, which are central actors instress situations. TheNAD is thus one of the main determinants of plant energy homeostasis,and therefore of cropyield. Through a reverse genetics approach targeting the L-aspartateoxidase (AO) – the firstcommitted enzyme of NAD biosynthesis – plants with constitutive eitherincreased ordecreased levels of pyridine nucleotides have been obtained. A furtherstudy of these plantsenabled to show that the NAD (1) can improve the growth, development andproductivity ofplants, (2) coordinates photosynthetic, respiratory and nitrogenmetabolisms, and (3) actsupon biotic and abiotic stress resistance mechanisms. This research isbeing valuedindustrially

Redox regulation of enzymes involved in sulfate assimilation and in the synthesis of sulfur-containing amino acids and glutathione in plants

International audienceSulfur is essential in plants because of its presence in numerous molecules including the two amino acids, cysteine, and methionine. Cysteine serves also for the synthesis of glutathione and provides sulfur to many other molecules including protein cofactors or vitamins. Plants absorb sulfate from their environment and assimilate it via a reductive pathway which involves, respectively, a series of transporters and enzymes belonging to multigenic families. A tight control is needed to adjust each enzymatic step to the cellular requirements because the whole pathway consumes energy and produces toxic/reactive compounds, notably sulfite and sulfide. Glutathione is known to regulate the activity of some intermediate enzymes. In particular, it provides electrons to adenosine 5′-phosphosulfate reductases but also regulates the activity of glutamate-cysteine ligase by reducing a regulatory disulfide. Recent proteomic data suggest a more extended post-translational redox control of the sulfate assimilation pathway enzymes and of some associated reactions, including the synthesis of both sulfur-containing amino acids, cysteine and methionine, and of glutathione. We have summarized in this review the known oxidative modifications affecting cysteine residues of the enzymes involved. In particular, a prominent regulatory role of protein persulfidation seems apparent, perhaps because sulfide produced by this pathway may react with oxidized thiol groups. However, the effect of persulfidation has almost not yet been explored

Evaluation and application of a targeted SPE-LC-MS method for quantifying plant hormones and phenolics in Arabidopsis

Application of metabolomics techniques to plant physiology is now considerable, and LC-MS is often being used for non-targeted, semi-quantitative analysis of effects caused by mutations or environmental conditions. However, examination of signalling metabolites like hormones require absolute rather than semi-quantitative quantitation, since their effect in planta is strongly dependent upon concentration. Further, plant hormones belong to different chemical classes and thus simultaneous quantitation remains highly challenging. Here we present an LC-MS method that allows the simultaneous absolute quantitation of six hormone families as well as selected phenolics. The technique requires solid phase extraction with a sulfonated cation exchange phase before analysis, and use calibration curves instead of isotopically labelled standards, which are indeed not commercially available for many hormonal molecules. The use of the total signal (including adducts) rather than a single quantifying mass appears to be crucial to avoid quantification errors because the ion distribution between adducts is found to be concentration-dependent. The different hormones considered appear to have contrasted ionisation efficiency due to their physical properties. However, the relatively low variability and the satisfactory response to standard additions show that the technique is accurate and reproducible. It is applied to Arabidopsis plants subjected to water stress, using either the wild-type or lines with altered NAD biosynthesis causing changes in salicylate signalling and phenylpropanoid levels. As expected, analyses show an increase in abscisic acid upon water stress and a consistent modification of phenolic compounds (including salicylate) in mutants

Characterization of l-aspartate oxidase from <em>Arabidopsis thaliana.</em>

UMR BFP - Equipe MétabolismeInternational audienceThe flavoprotein l-aspartate oxidase (LASPO) is the first enzyme of the de novo biosynthetic pathway of NAD+ in plants. Although LASPO is considered pivotal to maintain NAD+ homeostasis, it has not been hitherto characterized in plants. Here, the cDNA encoding the LASPO from the model plant Arabidopsis thaliana (AtLASPO, At5g14760) has been cloned and expressed in Escherichia coli for subsequent enzyme characterization. The purified AtLASPO enzyme displayed a Km of 0.79 mM for l-aspartate and a kcat of 0.25 s−1. We could further detect an l-aspartate: fumarate oxidoreductase activity of the recombinant plant enzyme. In addition, results indicated that NADP+ but not NAD+, and even more strongly NADH, inhibited AtLASPO at physiological concentrations by competing with the flavin for binding to the apoprotein. LASPO optimal pH and temperature, as well as plastidial pyridine nucleotide concentrations may contribute to an increased NAD+ production in planta. Moreover, in Arabidopsis thaliana AtLASPO gene expression exhibited a clear correlation between LASPO activity and NAD+ levels, thus demonstrating that plant LASPO catalyzes a key metabolic step of NAD+ synthesis