Modelling ozone stomatal flux over wheat under Mediterranean conditions

Correct estimation of leaf-level stomatal conductance (gsto) is central for current ozone (O3) risk assessment of wheat yield loss based on the absorbed O3 phytotoxic dose (POD). The gsto model parameterizations developed in Europe must be checked in the different climatic regions where they are going to be applied in order to reduce the uncertainties associated with the POD approach. This work proposes a new gsto model parameterization for estimating POD of Triticum aestivum and Triticum durum under Mediterranean conditions, based on phenological observations over 25 years and gsto field measurements during 5 growing seasons. Results show that POD in the Mediterranean area might be higher than previously estimated. However, caution must be paid when assessing the risk of yield loss for wheat in this area since field validation of O3 impacts is still limited.

Unlocking the resistance to wheat lipid transfer protein

Unlocking the resistance to wheat lipid transfer protein

In vitro ruminal fermentation of diets containing wheat straw and date pits as forage.

The pH, VFA concentration, total gas and met hane production were determined in the rumen of four Sicilo- Sarde rams fitted with permanent canulas. Rams received a ration that included 1.5 kg DM of oat hay and were supplemented with one of four concentrates: CC (10% barley, 43.3% corn, 25% wheat bran, 17.7% soybean meal, 4% sheep Vitamin and Mineral Mixture (VMM)), SC (66% white sorghum, 30% faba, 4% sheep VMM); TC (71% triticale, 18% faba, 7%, soybean meal, 4% VMM) or BC (71.5% barley, 17.5% faba, 7% soybean meal and 4% VMM). 50 ml samples were taken before, 2, 5 and 8 hours after the morning meal. Total gas was determined on rumen content before the morning meal. The rumen pH was statistically different (P<0.05) before and 2 hours after the morning meal among concentrates feed. It was in favour of TC and BC (P<0.05) concentrates but was comparable at the end of the day. The concentration of VFA was significantly higher (P<0.05) for diets TC and BC following the meal and became comparable among concentrates thereafter. The proportion of acetate and butyrate acids evolved in the same way during the day regardless of the regimen. The total volu me of gas was different (P<0.05) among diets, the BC showed the highest value (87.00±17.29 ml) while the lowest value was found in the TC concentrate (56.58±13.06 ml). The CH4 production for the BC was significantly different (P<0.05) from that of TC. Quantities produced by the CC and SC were similar (22.08±4.18vs . 21.16±3.21).

Component-resolved diagnosis of wheat flour allergy in baker's asthma.

Baker's asthma is one of the most common types of occupational asthma and its prevalence is increasing in the last years. Diagnosis of occupational asthma is complex. The poor specificity of current diagnostic approaches may be associated with insufficient purity of wheat extracts or lack of inclusion of major allergens in them. In this work, we use microarray technology to characterize the allergenic profiles of baker's asthma patients from three regions in Spain and to analyze the influence of other environmental allergens on the sensitization pattern.

Creation and Validation of the Spanish Durum Wheat Core Collection.

Spanish wheat (Triticum spp.) landraces have a considerable polymorphism, containing many unique alleles, relative to other collections. The existence of a core collection is a favored approach for breeders to efficiently explore novel variation and enhance the use of germplasm. In this study, the Spanish durum wheat (Triticum turgidum L.) core collection (CC) was created using a population structure–based method, grouping accessions by subspecies and allocating the number of genotypes among populations according to the diversity of simple sequence repeat (SSR) markers. The CC of 94 genotypes was established, which accounted for 17% of the accessions in the entire collection. An alternative core collection (CH), with the same number of genotypes per subspecies and maximizing the coverage of SSR alleles, was assembled with the Core Hunter software. The quality of both core collections was compared with a random core collection and evaluated using geographic, agromorphological, and molecular marker data not previously used in the selection of genotypes. Both core collections had a high genetic representativeness, which validated their sampling strategies. Geographic and agromorphological variation, phenotypic correlations, and gliadin alleles of the original collection were more accurately depicted by the CC. Diversity arrays technology (DArT) markers revealed that the CC included genotypes less similar than the CH. Although more SSR alleles were retained by the CH (94%) than by the CC (91%), the results showed that the CC was better than CH for breeding purposes.

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterización molecular de BdDOF24 durante la germinación de las semillas

La semilla es el órgano que garantiza la propagación y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentación humana y animal. La semilla de cereales acumula en el endospermo durante la maduración, mayoritariamente, almidón y proteínas de reserva. Estas reservas son hidrolizadas en la germinación por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energía hasta que la plántula emergente es fotosintéticamente activa. Ambas fases del desarrollo de la semilla, están reguladas por una red de factores de transcripción (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son proteínas que han desempeñado un papel central en la evolución y en el proceso de domesticación, siendo uno de los principales mecanismos de regulación génica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de unión a DNA, éstos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias proteínas DOF que desempeñan un papel fundamental en la regulación de la expresión génica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su pequeño tamaño, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (plásmido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agronómica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un árbol filogenético en base al alineamiento múltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El análisis filogenético establece cuatro grupos de genes ortólogos (MCOGs: Major Clusters of Orthologous Genes), que están validados por motivos conservados adicionales, además del dominio DOF, entre las secuencias de las proteínas de un mismo MCOG. El estudio global de expresión en diferentes órganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresión de estos genes durante la maduración y germinación muestra que BdDof24, ortólogo putativo a BPBF-HvDOF24 de cebada, es el gen más abundante en las semillas en germinación de B. distachyon. La regulación transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post‐germinación ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ortólogo a los genes Al21 de trigo y HvCathB de cebada, así como los TFs responsables de su regulación transcripcional BdDOF24 y BdGAMYB (ortólogo a HvGAMYB). El análisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posición y secuencia, con sus ortólogos en trigo y cebada. La expresión de BdCathB se induce durante la germinación, así como la de los genes BdDof24 y BdGamyb. Además, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos híbridos de levadura e in planta en experimentos de complementación bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ortólogos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las células de aleurona se transforman simultáneamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activación mediada por BdGAMYB, mientras que su ortólogo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las proteínas BdDOF24 y BPBF-HvDOF24 podrían explicar las funciones opuestas que desempeñan en su interacción con GAMYB. Resultados preliminares con líneas de inserción de T-DNA y de sobre-expresión estable de BdGamyb, apoyan los resultados obtenidos en expresión transitoria. Además las líneas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Application of Improved Direct Calibration for Hyperspectral image processing: Detecting peanut traces in wheat flour.

NIR Hyperspectral imaging (1000-2500 nm) combined with IDC allowed the detection of peanut traces down to adulteration percentages 0.01% Contrary to PLSR, IDC does not require a calibration set, but uses both expert and experimental information and suitable for quantification of an interest compound in complex matrices. The obtained results shows the feasibility of using HSI systems for the detection of peanut traces in conjunction with chemical procedures, such as RT-PCR and ELISA

Detection of peanut traces in wheat flour through NIR hyperspectral imaging spectroscopy

- NIR Hyperspectral images (1000-2200 nm) allowed the detection of peanut traces down to adulteration percentages 0.01 % - Determination coefficient of R2= 0.946 was found for the quantification of peanut adulteration from 10% to 0.1%. - The obtained results shows the feasibility of using HSI systems for the detection of peanut traces in conjuction with chemical procedures, such as RT-PCR and ELISA to facilitate quality control surveyance on food product processing lines.

Growth rate and TRI5 gene expression profiles of Fusarium equiseti strains isolated from Spanish cereals cultivated on wheat and barley media at different environmental conditions

Fusarium equiseti is a toxigenic species that often contaminates ce real crops from diverse climatic regions such as Northern and Southern Europe. Previous results suggested the existence of two distinct populations within this species with differences in toxin pro file which largely corresponded to North and South Europe (Spain). In this work, growth rate profiles of 4 F. equiseti strains isolated from different cereals and distinct Spanish regions were determined on wheat and barley based media at a range of temperatures (15, 20, 25, 30, 35 and 40 °C) and water potentialregimens(−0.7,−2.8,−7.0,and −9.8MPa,correspondingto 0.99,0.98,0.95 and 0.93aw values).Growth was observed at all temperatures except at 40 °C, and at all the solute potential values except at−9.8 MPa when combined with 15 °C. Optimal growth was observed at 20– 30 °C and −0.7/−2.8 MPa. The effect of these factors on trichothecene biosynthesis was examined on a F. equiseti strain using a newly developed real time RT-PCR protocol to quantify TRI5 gene expression at 15, 25 and 35 °C and −0.7, −2.8, − 7.0 and −9.8 MPa on wheat and barley based media. Induction of TRI5 expression was detected between 25 and 35 °C and −0.7 and − 2.8 MPa, with maximum values at 35 °C and −2.8 MPa being higher in barley than in wheat medium. These results appeared to be consistent with a population well adapted to the present climatic conditions and predicted scenarios for Southern Europe and suggested some differences depending on the cereal considered. These are also discussed in relation to other Fusarium species co-occurring in cereals grown in this region and to their significance for prediction and control strategies of toxigenic risk in future scenarios of climate change for this region.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Short communication. Collection and characterisation of a population of Triticum boeoticum Boiss., a wild wheat species not previously found in the Mediterranean western region

En julio del 2010 se recolectó una población de trigo silvestre en una zona abandonada cerca de Madrid, España. Esta zona posee una biodiversidad botánica elevada y un tipo de suelo muy peculiar denominado “arcillas verdes”. Se recogió una muestra de trigo y se multiplicó y caracterizó para varios caracteres agro-morfológicos y subunidades de gluteninas. El número cromosómico 2n de las semillas demostró que es una especie diploide de trigo y los datos de caracterización indicaron que es Triticum boeoticum Boiss. Esta especie llegó probablemente como mala hierba del cultivo de escaña que se producía en la zona hasta al menos la primera mitad del s. xix. Las características edáficas y climáticas del lugar y el hecho de que no haya referencias hasta ahora de esta especie en la zona oeste de la región Mediterránea aumentan el valor de esta adquisición para la mejora del trigo. La nueva accesión se conserva en el Centro Nacional de Recursos Fitogenéticos y se ha depositado una hoja de herbario en el Real Jardín Botánico de Madrid

Dietary xylanase increases hepatic vitamin E concentration of chickens fed wheat based diet

The study examined the effect of xylanase supplementation on apparent metabolizable energy (AME) and hepatic vitamin E and carotenoids in broiler chickens fed wheat based diets. A total of one hundred forty four male Ross 308 chickens were used in this study. Birds were randomly assigned to 3 dietary treatments (8 cages per treatment of 6 male broilers each) for 14 days from 7 to 21 day old. The control treatment was based on wheat-soyabean meal and was either unsupplemented or supplemented with either 1000 or 2000 xylanase units per kg diet. Orthogonal polynomial contrasts were used to test linear response to dietary xylanase activity. There was a positive linear relationship (P < 0.05) between dietary AME and doses of supplementary xylanase. A linear relationship (P < 0.05) was also observed between dosage of xylanase supplementation and hepatic vitamin E concentration and retention. In conclusion, xylanase supplementation improved dietary AME and increased hepatic vitamin E concentration which may have positive effects on the antioxidative status of the birds.

Partioning of dietary energy of chickens fed maize or wheat-based diets with and without a commercial blend of phytogenic feed additives

The aim of the study was to investigate the effects of a standardized mixture of a commercial blend of phytogenic feed additives containing 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum on utilization of dietary energy and performance in broiler chickens. Four experimental diets were offered to the birds from 7 to 21 d of age. These included 2 basal control diets based on either wheat or maize that contained 215 g CP/kg and 12.13 MJ/kg ME and another 2 diets using the basal control diets supplemented with the plant extracts combination at 100 mg/kg diet. Each diet was fed to 16 individually penned birds following randomization. Dietary plant extracts improved feed intake and weight gain (P < 0.05) and slightly (P < 0.1) improved feed efficiency of birds fed the maize-based diet. Supplementary plant extracts did not change dietary ME (P > 0.05) but improved (P < 0.05) dietary NE by reducing the heat increment (P < 0.05) per kilogram feed intake. Feeding phytogenics improved (P < 0.05) total carcass energy retention and the efficiency of dietary ME for carcass energy retention. The number of interactions between type of diet and supplementary phytogenic feed additive suggest that the chemical composition and the energy to protein ratio of the diet may influence the efficiency of phytogenics when fed to chickens. The experiment showed that although supplementary phytogenic additives did not affect dietary ME, they caused a significant improvement in the utilization of dietary energy for carcass energy retention but this did not always relate to growth performance.

Hyperspectral to multispectral imaging for detection of tree nuts and peanut traces in wheat flour

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and compared with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

Detection and quantification of peanut traces in wheat flour by near infrared hyperspectral imaging spectroscopy using principal-component analysis

The use of a common environment for processing different powder foods in the industry has increased the risk of finding peanut traces in powder foods. The analytical methods commonly used for detection of peanut such as enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) represent high specificity and sensitivity but are destructive and time-consuming, and require highly skilled experimenters. The feasibility of NIR hyperspectral imaging (HSI) is studied for the detection of peanut traces down to 0.01% by weight. A principal-component analysis (PCA) was carried out on a dataset of peanut and flour spectra. The obtained loadings were applied to the HSI images of adulterated wheat flour samples with peanut traces. As a result, HSI images were reduced to score images with enhanced contrast between peanut and flour particles. Finally, a threshold was fixed in score images to obtain a binary classification image, and the percentage of peanut adulteration was compared with the percentage of pixels identified as peanut particles. This study allowed the detection of traces of peanut down to 0.01% and quantification of peanut adulteration from 10% to 0.1% with a coefficient of determination (r2) of 0.946. These results show the feasibility of using HSI systems for the detection of peanut traces in conjunction with chemical procedures, such as RT-PCR and ELISA to facilitate enhanced quality-control surveillance on food-product processing lines.