Prolamin content and grain weight in RNAi silenced wheat lines under different conditions of temperature and nitrogen availability

Temperature and nitrogen (N) availability are two important environmental factors that may produce important changes in grain composition during grain filling of bread wheat. In this study, four wheat lines with the down-regulation of gliadins by means of RNA interference (RNAi) have been characterized to determine the effect of thermal stress and N availability on grain weight and quality; with focus on gliadin and glutenin protein fractions. Grain weight was reduced with heat stress (HS) in all RNAi lines, whereas gliadin content was increased in the wild-types. With respect to gliadin content, RNAi lines responded to HS and N availability differently from their respective wild-types, except for ω-gliadin content, indicating a very clear stability of silencing under different environmental conditions. In a context of increased temperature and HS events, and in environments with different N availability, the RNAi lines with down-regulated gliadins seem well suited for the production of wheat grain with low gliadin content.The Spanish Ministry of Economy and Competitiveness (Project AGL2016-80566-P) and the European Regional Development Fund (FEDER) supported this research

Characterization of Changes in Gluten Proteins in Low-Gliadin Transgenic Wheat Lines in Response to Application of Different Nitrogen Regimes

Gluten proteins are major determinants of the bread making quality of wheat but also of important gluten-related disorders. The gluten protein accumulation during grain filling is strongly influenced by nitrogen fertilization. We have characterized the gluten proteins in low-gliadin wheat lines as influenced by nitrogen treatments in two experiments. These transgenic lines, D783, D793, C655, D577, and E82 were obtained by using two different RNAi silencing fragments and two endosperm-specific promoters to drive the silencing fragments (d-hordein and γ-gliadin). In Experiment 1, we used three nitrogen fertilizer rates (120, 360, and 1080 mg N) added at sowing stage and combined with two sulfur rates (8 and 30 mg S); Experiment 2 included two nitrogen levels (120 and 1080 mg N), which were added according to the greatest demand per plant using split applications. The protein quantification was accomplished by Reverse-Phase High-Performance Liquid Chromatography and gluten content (ppm) determined using monoclonal antibody R5 (Competitive R5 ELISA). The results showed differences in protein accumulation between the two transgenic lines with the same silencing fragment but different promoter. Lines D793 and E82 showed low gliadin and an increment in glutenin content with increasing nitrogen. Competitive ELISA R5 showed a significant decrease in gluten content using split applications of nitrogen (Experiment 2) with 120 mg N compared to Experiment 1. In addition, line E82 ensures that variations in N fertilization will not result in increased gluten content.The Spanish Ministry of Economy and Competitiveness (Projects AGL2013-48946-C3-1-R and AGL2016-80566-P), the European Regional Development Fund (FEDER) and Junta de Andalucía (Project P11-AGR-7920) supported this work.Peer Reviewe

Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley-wheat amphiploids

<p>Abstract</p> <p>Background</p> <p>The <it>APETALA2</it>-like genes form a large multi-gene family of transcription factors which play an important role during the plant life cycle, being key regulators of many developmental processes. Many studies in <it>Arabidopsis </it>have revealed that the <it>APETALA2 </it>(<it>AP2</it>) gene is implicated in the establishment of floral meristem and floral organ identity as well as temporal and spatial regulation of flower homeotic gene expression.</p> <p>Results</p> <p>In this work, we have cloned and characterised the <it>AP2</it>-like gene from accessions of <it>Hordeum chilense </it>and <it>Hordeum vulgare</it>, wild and domesticated barley, respectively, and compared with other <it>AP2 </it>homoeologous genes, including the Q gene in wheat. The <it>Hordeum AP2</it>-like genes contain two plant-specific DNA binding motifs called AP2 domains, as does the Q gene of wheat. We confirm that the <it>H. chilense AP2</it>-like gene is located on chromosome 5H^ch. Patterns of expression of the <it>AP2</it>-like genes were examined in floral organs and other tissues in barley, wheat and in tritordeum amphiploids (barley × wheat hybrids). In tritordeum amphiploids, the level of transcription of the barley <it>AP2</it>-like gene was lower than in its barley parental and the chromosome substitutions 1D/1H^chand 2D/2H^chwere seen to modify <it>AP2 </it>gene expression levels.</p> <p>Conclusion</p> <p>The results are of interest in order to understand the role of the <it>AP2</it>-like gene in the spike morphology of barley and wheat, and to understand the regulation of this gene in the amphiploids obtained from barley-wheat crossing. This information may have application in cereal breeding programs to up- or down-regulate the expression of <it>AP2</it>-like genes in order to modify spike characteristics and to obtain free-threshing plants.</p

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante iARN

Peer reviewedConsejo Superior de Investigaciones CientíficasT3 Traducción de patente europe

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi. La presente invención se refiere al silenciamiento especifico de las (alfa), (beta), (gamma) y ! (omega)- gliadinas de trigo duro y harinero mediante RNA de interferencia (ARNi) por medio del empleo de un polinucleótido que se transcribe a un hpRNA (hairpin RNA). Además, la presente invención también se refiere a un vector, célula, planta o semilla que comprenden el polinucleótido, cuya expresión se dirige de forma específica en tejidos concretos de las semillas de trigo mediante secuencias reguladoras de la expresión génica como por ejemplo, el promotor de un gen de -gliadinas o el promotor del gen que codifica para una D-hordeína.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Down-Regulating γ-gliadins in bread wheat leads to non-specific increases in other gluten proteins and has no major effect on dough gluten strength.

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten protein

Integrating granular activated carbon in the post-treatment of membrane and settler effluents to improve organic micropollutants removal

Granular activated carbon (GAC) is applied as post-treatment technology in wastewater treatment plants (WWTPs) in order to increase the elimination of organic micropollutants (OMPs). However, the efficiency and life-time of GAC depend on several parameters, such as the quality of the effluent to be treated or the type of GAC. In the present paper, two types of GAC, based on bituminous carbon (BC-GAC) and coconut shell (CS-GAC), were assessed from a technical, economic and environmental point of view to further remove OMPs present in two secondary effluents, coming from integrated biological systems with a membrane or a settler, respectively. Although all GAC filters were efficient in removing selected OMPs, the quality of the secondary effluent had a strong influence on the lifespan of adsorbent material and the technical operability of the filtration systems. While GAC filters treating membrane effluent were highly effective to remove recalcitrant compounds, such as carbamazepine and diazepam (>80%), even after 430 d of operation (>30,800 BV), the efficiency of GAC filters treating settler effluent quickly lowered to 50% after 100 d of operation (<7200 BV). Both types of GAC showed similar adsorption capacities and only slight differences were found in terms of costs (2.4 €/kg vs 2.7 €/kg). However, CS-GAC has a lower carbon footprint than BC-GAC, mainly due to the more environmentally friendly production process of CS-GACThis research was supported by the Spanish Government (AEI – Spain) through the projects HOLSIA (CTM2013-46750-R) and COMETT (CTQ2016-80847-R). The authors belong to the Galician Competitive Research Group GRC ED431C2017/29 and to the CRETUS Strategic Partnership (AGRUP2015/02). All these programmes are co-funded by FEDER (EU)S

Glyphosate-Resistant Parthenium hysterophorus in the Caribbean Islands: Non Target Site Resistance and Target Site Resistance in Relation to Resistance Levels

Peer reviewedGlyphosate has been the most intensely herbicide used worldwide for decades, and continues to be a single tool for controlling weeds in woody crops. However, the adoption of this herbicide in a wide range of culture systems has led to the emergence of resistant weeds. Glyphosate has been widely used primarily on citrus in the Caribbean area, but a study of resistance in the Caribbean islands of Cuba and the Dominican Republic has never been carried out. Unfortunately, Parthenium hysterophorus has developed glyphosate-resistance in both islands, independently. The resistance level and mechanisms of different P. hysterophorus accessions (three collected in Cuba (Cu-R) and four collected in the Dominican Republic (Do-R) have been studied under greenhouse and laboratory conditions. In in vivo assays (glyphosate dose causing 50% reduction in above-ground vegetative biomass and survival), the resistance factor levels showed susceptible accessions (Cu-S ≥ Do-S), low-resistance accessions (Cu-R3 Do-R2 > Cu-R2 > Do-R3 > Do-R4 > Cu-R3 >> Cu-S ≥ Do-S). Glyphosate was degraded to aminomethylphosphonic acid, glyoxylate and sarcosine by >88% in resistant accessions except in Cu-R3 and Do-R4 resistant accessions (51.12 and 44.21, respectively), whereas a little glyphosate (<9.32%) was degraded in both susceptible accessions at 96 h after treatment. There were significant differences between P. hysterophorus accessions in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with and without different glyphosate rates. The R accessions showed values of between 0.026 and 0.21 μmol μg−1 TSP protein min−1 basal EPSPS activity values with respect to the S (0.024 and 0.025) accessions. The same trend was found in the EPSPS enzyme activity treated with glyphosate, where a higher enzyme activity inhibition (glyphosate μM) corresponded to greater resistance levels in P. hysterophorus accessions. One amino acid substitution was found at position 106 in EPSPS, consisting of a proline to serine change in Cu-R1, Do-R1 Do-R2. The above-mentioned results indicate that high resistance values are determined by the number of defense mechanisms (target-site and non-target-site resistance) possessed by the different P. hysterophorus accessions, concurrently.This work was funded by AGL2013-48946-C3-1-R and AGL2016-78944-R projects (Spain).Peer Reviewe

Validation of Cod maturity ogive in NAFO 3M. Effect of the reproductive ccycle moment on ogive estimation.

The Flemish Cap (FC) survey is the source of the Atlantic cod maturity data for SSB estimation. FC survey takes place when females are resting and discrimination between spawning active and inactive females is still possible testing the presence of postovulatory follicles, that remains several months after spawning. However, the best moment for discriminate active spawning females is during the spawning season. For 2012 and 2014, the maturity ogives calculated during the spawning season and when females are resting were compared. Assuming a seasonal growth for cod in 3M, the moment in which maturity is estimated, had no effect on the ogives estimation. Thus, it is recommended to continue employing in the 3M cod assessment the maturity ogives produced with the FC survey information

Characterization of celiac disease related oat proteins: bases for the development of high quality oat varieties suitable for celiac patients

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients.España Ministerio de Economía y Competitividad Project IPT-2011-1321-010000España, Junta de Andalucía Project P12-AGR-176