Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice.

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.

Neuroscience robotics to investigate multisensory integration and bodily awareness.

Humans experience the self as localized within their body. This aspect of bodily self-consciousness can be experimentally manipulated by exposing individuals to conflicting multisensory input, or can be abnormal following focal brain injury. Recent technological developments helped to unravel some of the mechanisms underlying multisensory integration and self-location, but the neural underpinnings are still under investigation, and the manual application of stimuli resulted in large variability difficult to control. This paper presents the development and evaluation of an MR-compatible stroking device capable of presenting moving tactile stimuli to both legs and the back of participants lying on a scanner bed while acquiring functional neuroimaging data. The platform consists of four independent stroking devices with a travel of 16-20 cm and a maximum stroking velocity of 15 cm/s, actuated over non-magnetic ultrasonic motors. Complemented with virtual reality, this setup provides a unique research platform allowing to investigate multisensory integration and its effects on self-location under well-controlled experimental conditions. The MR-compatibility of the system was evaluated in both a 3 and a 7 Tesla scanner and showed negligible interference with brain imaging. In a preliminary study using a prototype device with only one tactile stimulator, fMRI data acquired on 12 healthy participants showed visuo-tactile synchrony-related and body-specific modulations of the brain activity in bilateral temporoparietal cortex.

Looking for factors involved in the light-dependent degradation of phytochrome A

SUMMARY : Phytochromes constitute a family of red/far-red photoreceptors regulating all the major transitions during the life cycle of plants. In Arabidopsis, five members: phyA,_ B, C, D and E, were identified. Phytochromes are synthesized in their inactive red-light absorbing form called Pr. Upon light absorbance they convert to the far-red light absorbing Pfr form. The Pfr form is the active conformer which converts back to the Pr form either rapidly upon far-red perception or in a slower process called dark reversion. ph~A represents an exception, in that it does not significantly dark-revert and two specific processes have been developed by the plants to decrease the amount of biologically active phyA. The first one is alight-dependent repression of the PHYA gene expression and the second one is alight-dependent degradation of the phyA protein. The latter is the most efficient process to rapidly decrease the level of active phyA. The ability of plants to regulate the amount of active phyA is critical in a far-red rich environment, a situation observed under a canopy. In these conditions, phyA is essential to induce the germination and the deetiolation of the young seedling. Later in the development the ability of phyA to repress growth counteracts the shade avoidance response. Therefore decreasing the amount of phyA allows stem growth and to compete with neighbours for the light. In this thesis, I investigate the light-dependent degradation of phyA. I developed a reverse genetic approach based on the systematic analysis of the light-dependent accumulation of phyA in the different cullin mutant cull, cul3a; cul3b and cul4. This analysis allowed me to show that CUL1 and CUL3A-based E3 ligase complexes are involved in the regulation of phyA degradation. Surprisingly, our results also demonstrate that cu14 is not affected in the degradation of phyA whereas constitutive Photomorphogenic 1 (COP1) a subunit of one CUL4based E3 complex was reported to be involved. Further investigations showed that the phenotype of cop1 is conditional, the mutant being defective in phyA degradation only in the presence of metabolisable sugars. I also showed that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and in the nucleus using mutants and transgenic lines affected in the localization of phyA. Interestingly, I observed that phyA degradation was faster in the nucleus than in the cytosol and that rapid degradation of Pr also occurred in the nucleus suggesting that cytosolic accumulation of phyA in the dark is a way to regulate its proteolysis. Finally, we identify a short region similar to a PEST sequence required for phyA stability and we developed a unbiased genetic screen to identify new components involved in the regulation of the light-dependent degradation of phyA. The significance of these results are discussed. RESUME : Les phytochromes (phy) constituent une famille de photorécepteurs absorbant la lumière rouge et rouge lointaine et régulant toutes les étapes de transitions majeures dans la vie des plantes. Chez Arabidopsis, cinq membres : phyA, B, C, D et E ont été identifiés. Les phytochromes sont synthétisés sous une forme inactive appelée Pr absorbant la lumière rouge. Après perception de lumière ils passent sous une forme active Pfr absorbant dans le rouge lointain. La forme Pfr peut retourner sous la forme Pr après absorption de lumiëre rouge lointaine ou dans un processus lent appelé «réversion à l'obscurité ». phyA représente une exception à cette règle car il ne retoune pas significativement sous sa forme inactive dans le noir. Deux processus spécifiques ont donc été développés pour diminuer le taux de phyA actif. Le premier consiste en la répression du gène PHYA en condition de lumière et le second en une dégradation induite par la lumière de la protéine phyA. Ce dernier processus est le plus efficace pour diminuer rapidement le niveau de phyA. La capacité des plantes à réguler le taux de phyA actifs est critique dans un environnement riche en lumière rouge lointaine, une situation observée sous une canopée. Sous une canopée, phyA est essentiel pour induire la germination et la dé-étiolation de la jeune pousse. Plus tard dans le développement la capacité de phyA de réprimer la croissance freine la «réponse à l'évitement de l'ombre ». Par conséquent diminuer le taux de phyA permet la croissance de la tige et donc de rentrer en compétition pour la lumière avec les plantes avoisinantes. Dans cette thèse, j'ai étudié la dégradation de phyA. J'ai développé une approche génétique inverse basée sur l'analyse systématique de l'accumulation de phyA en condition de lumière dans les différents mutants cullin, cul1, cul3a, cul3b et cul4. Ces analyses nous ont permis d'identifier qu'un complexe E3 ligase CUL1 et un complexe E3 ligase CUL3A sont impliqués dans la régulation de la dégradation de phyA. Mes résultats démontrent aussi que le mutant cul4 n'est pas affecté dans la dégradation de phyA alors que Çonstitutive Photomorphogenic 1 (COPI) une sous unité d'un complexe CUL4 à été identifier dans la régulation de cette dégradation. Des analyses supplémentaires suggèrent que l'effet de la mutation cop1 est dépendante dë la présence de sucres métabolisables. J'ai aussi montré que phyA est dégradé dans le noyau et dans le cytoplasme par un mécanisme dépendant du protéasome et que la dégradation dans le.noyau est non seulement aspécifique de la forme Pr ou Pfr mais aussi est plus rapide que dans le cytoplasme. Ceci suggère que l'accumulation de phyA dans le cytoplasme permet son accumulation à des niveaux élevés à l'obscurité. Enfin j'ai identifié une région similaire à un motif PEST requise pour la stabilité de phyA et j'ai aussi développé un criblage génétique non biaisé pour identifier de nouveaux composants impliqués dans la régulation de la dégradation de phyA. L'importance de ces résultats est discutée dans le dernier chapitre de cette thèse.

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Le "neurally adjusted ventilatory assist": vers une révolution de la ventilation mécanique [Neurally adjusted ventilatory assist: a revolution of mechanical ventilation?].

Neurally adjusted ventilatory assist or NAVA is a new assisted ventilatory mode which, in comparison with pressure support, leads to improved patient-ventilator synchrony and a more variable ventilatory pattern. It also improves arterial oxygenation. With NAVA, the electrical activity of the diaphragm is recorded through a nasogastric tube equipped with electrodes. This electrical activity is then used to pilot the ventilator. With NAVA, the patient's respiratory pattern controls the ventilator's timing of triggering and cycling as well as the magnitude of pressurization, which is proportional to inspiratory demand. The effect of NAVA on patient outcome remains to be determined through well-designed prospective studies.

Entomopathogenic fungal (Hyphomycetes) collection: assessment of conidial viability

Twenty four strains of the entomopathogenic fungi (Hyphomycetes) Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, P. fumosoroseus and P. lilacinus, maintained in the culture collection of Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen) and preserved by lyophilization and in liquid nitrogen, had their conidial viability assessed. Germination rates of 16- to 84-month-old cultures stored in liquid nitrogen decreased, on average, less than 13.3%. For 29- to 49-month-old cultures preserved by lyophilization, the viability loss ranged, on average, from 28.6 to 94.5%. The results demonstrated the efficiency of the tested methods, especially liquid nitrogen, in preserving the viability of entomopathogenic fungi.

Soil solarization for weed control in carrot

Soil solarization is a technique used for weed and plant disease control in regions with high levels of solar radiation. The effect of solarization (0, 3, 6, and 9 weeks) upon weed populations, carrot (Daucus carota L. cv. Brasília) yield and nematode infestation in carrot roots was studied in São Luís (2º35' S; 44º10' W), MA, Brazil, using transparent polyethylene films (100 and 150 mm of thickness). The maximum temperature at 5 cm of depth was about 10ºC warmer in solarized soil than in control plots. In the study 20 weed types were recorded. Solarization reduced weed biomass and density in about 50% of weed species, including Cyperus spp., Chamaecrista nictans var. paraguariensis (Chod & Hassl.) Irwin & Barneby, Marsypianthes chamaedrys (Vahl) O. Kuntze, Mitracarpus sp., Mollugo verticillata L., Sebastiania corniculata M. Arg., and Spigelia anthelmia L. Approximately 40% of species in the weed flora were not affected by soil mulching. Furthermore, seed germination of Commelina benghalensis L. was increased by soil solarization. Marketable yield of carrots was greater in solarized soil than in the unsolarized one. It was concluded that solarization for nine weeks increases carrot yield and is effective for controlling more than half of the weed species recorded. Mulching was not effective for controlling root-knot nematodes in carrot.

Nonenzymatic lipid peroxidation reprograms gene expression and activates defense markers in Arabidopsis tocopherol-deficient mutants.

Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Avaluació de l'aptitud de bioestabilitzats com a esmena orgànica per a la rehabilitació de sòls

L’objectiu d’aquest estudi ha estat avaluar l’efecte que té sobre algunes propietats fisicoquímiques del sòl i el creixement de plantes, l’adició del producte compostat obtingut a partir de la fracció orgànica dels residus sòlids urbans de recollida no selectiva, material anomenat bioestabilitzat. S’ha dut a terme un bioassaig amb blat (Triticum aestivum), utilitzant bioestabilitzat procedent de dues plantes de tractament mecànic i biològic, Vacarisses i Mataró, que s’ha aplicat a diferents dosis en un sòl de baixa qualitat. S’ha observat una relació positiva entre el creixement del blat i la dosis de bioestabilitzat aplicada. Pel que fa a la germinació, ha estat lleugerament estimulada per les dosis intermèdies de bioestabilitzat assajades. En quant a les propietats fisicoquímiques del sòl, s’ha observat un increment de la salinitat en funció de la dosis de bioestabilitzat aplicada, que en les més elevades podria ser problemàtica. També, el contingut de matèria orgànica ha augmentat de manera proporcional a la dosis, cosa que ha millorat les propietats físiques del sòl. Per altra banda, s’han trobat continguts d’impropis (vidre, plàstic ...) i concentracions d’alguns metalls pesants bastant elevades, així com una considerable variabilitat entre les partides de bioestabilitzat. Finalment, es conclou que abans de permetre l’aplicació de bioestabilitzat per a la rehabilitació de sòls degradats, cal aprofundir més en l’estudi d’aquest material.

EEG signal analysis via a cleaning procedure based on multivariate empirical mode decomposition

Artifacts are present in most of the electroencephalography (EEG) recordings, making it difficult to interpret or analyze the data. In this paper a cleaning procedure based on a multivariate extension of empirical mode decomposition is used to improve the quality of the data. This is achieved by applying the cleaning method to raw EEG data. Then, a synchrony measure is applied on the raw and the clean data in order to compare the improvement of the classification rate. Two classifiers are used, linear discriminant analysis and neural networks. For both cases, the classification rate is improved about 20%.

Embryo rescue from interspecific crosses in apple rootstocks

The objetive of this work was to rescue immature embryos of apple rootstocks Malus prunifolia (Marubakaido) and Malus pumila (M9) after 40-60 days of pollination and to put them into MS culture media supplemented with agar (6 g L-1) and casein hydrolysate (500 mg L-1). Embryos originated from interspecific crosses and open pollination showed differences in the in vitro responses, depending on the female parent, the developmental stage of the embryo, and the culture medium composition. Embryos of the M. pumila rootstock, rescued within 40 days after pollination and put in culture medium supplemented with indolacetic acid (IAA), gibberellic acid (GA3), kinetin and maltose, resulted in a normal development of plantlets. However, embryos originating from hand-pollination, cultivated in medium supplemented with 14 µM IAA, 5 µM kinetin and 1.5 µM Ga3 (MS1), mainly those of M. prunifolia x M. pumila, showed a high percentage of rusted embryos (96.2%). Embryos from open pollination of M. prunifolia and M. pumila formed calluses. It was possible to identify the influence of the female parent by the enhanced development of M. pumila shoots derived from open or hand-pollination. The crossing of responsive species and the use of the technique of embryo culture provided a rapid and uniform germination and, consequently, the development of fully normal seedlings.

Electrical conductivity testing of corn seeds as influenced by temperature and period of storage

The objective of this work was to evaluate the effects of temperature (10, 20, 30, 20/10 and 30/10ºC) and period of storage on electrical conductivity (EC) in four seed lots of corn (Zea mays L.), as well as the mineral composition of the soaking solution. EC test determines indirectly the integrity of seed membrane systems, and is used for the assessment of seed vigor, because this test detects the seed deterioration process since its early phase. The research comprised determinations of water content, germination, accelerated aging (AA), cold (CT) and EC vigor tests, and determinations of Ca2+, Mg2+ and K+ release to the solution, after seed soaking of four corn seed lots. The evaluations were performed each four months during a period of 16 months. For statistical analysis, a completely randomized split plot design was used with eight replications. Except for seed lots stored at 10ºC, all vigor evaluations revealed a decline in vigor, but AA and CT showed more sensitiveness to declines of seed physiological quality than EC. Potassium was the main leached ion regardless of the storage temperature.

Green manure incorporation timing for organically grown broccoli

The objective of this work was to determine the effect of incorporation timing of the velvet bean (Stizolobium cinereum) (GM) on both organic broccoli yield and N status. Mineral N content in the soil, biologically fixed N recovery by broccoli, GM biomass decomposition and N release kinetics were also determined. Plots were fertilized with 12 Mg ha-1 of organic compost and received GM either at 0, 15, 30 or 45 days after transplant. Other treatments were compost (12 or 25 Mg ha-1), GM, mineral fertilizers and control (no fertilizer). The data were collected in four completely randomized blocks. GM decomposition increased mineral N content in soil as rapidly as mineral fertilizer or the supply of 25 Mg ha-1 of compost. The N half-life in GM (24 days) is smaller than the mass half-life (35 days) and the biological fixation contributed with 23.6% of N present in the aboveground biomass of broccoli. The result suggests a higher synchrony between the crop relative growth rate and N release from the GM when incorporated at crop early growth stage. The incorporation of GM until 15 days after transplanting replaces 50% of the highest compost dose, without yield loss.