Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Caracterización molecular del mutante riso 1508 de cebada : modificaciones transcripcionales y posttranscripcionales en el desarrollo de la semilla

La semilla es el principal órgano reproductivo de las plantas espermatofitas, permitiendo la dispersión de las poblaciones y asegurando su supervivencia gracias a su tolerancia a la desecación y a su capacidad para germinar bajo condiciones ambientales óptimas. El rendimiento y valor económico de los cereales, que constituyen la primera cosecha mundial, depende, en buena medida, de la eficacia con que se acumulan en la semilla sustancias de reserva: proteínas, carbohidratos y lípidos. El principal carbohidrato acumulado en la semilla de cebada es el almidón y la fracción mayoritaria de proteínas es la de las prolaminas (solubles en etanol al 70%); estas proteínas tienen muy bajo contenido en lisina, un aminoácido esencial en la dieta de animales monogástricos. Con el fin de mejorar el valor nutricional de la semilla de cebada, se han obtenido diferentes mutantes con un mayor contenido en este aminoácido. Riso 1508 es un mutante de cebada rico en lisina cuya mutación lys3a, de efectos pleiotrópicos, segrega como un único gen mendeliano. Entre otros, presenta una reducción drástica de la expresión de algunos genes que codifican proteínas de reserva de tipo prolamina, en concreto, presenta reducida la expresión de los genes que codifican B-, C- y ϒ-Hordeínas y del inhibidor de tripsina CMe, pero no tiene alterada la expresión del gen que codifica las D-Hordeínas. Este último gen carece en su promotor del motivo GLM (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), que es reconocido por factores transcripcionales bZIP. En este trabajo, el mutante de cebada Riso 1508 se ha utilizado como herramienta para profundizar en el conocimiento de la regulación génica en semillas durante las fases de la maduración y la germinación. Para ello, en una primera aproximación, se llevó a cabo un análisis transcriptómico comparando el genotipo mutante con el silvestre durante la maduración de la semilla. Además de confirmar variaciones en los genes que codifican proteínas de reserva, este análisis indicó que también estaban afectados los genes relacionados con metabolismo de carbohidratos. Por ello se decidió caracterizar la familia multigénica de sacarosas sintasa (SUSy) en cebada. Se anotaron dos nuevos genes, HvSs3 y HvSs4, cuya expresión se comparó con la de los genes HvSs1 y HvSs2, previamente descritos en el laboratorio. La expresión de los cuatro genes en tejidos diferentes y su respuesta a estreses abióticos se analizó mediante RT-qPCR. HvSs1 y HvSs2 se expresaron preferencialmente durante el desarrollo del endospermo, y HvSs1 también fue un tránscrito abundante durante la germinación. HvSs1 se indujo en hojas en condiciones de anoxia y HvSs3 por estrés hídrico, y ambos genes se indujeron por tratamientos de frío. La localización subcelular de las cuatro isoformas no fue sólo citoplásmica, sino que también se localizaron en zonas próximas a retículo endoplásmico y en la cara interna de la membrana plasmática; además, se observó una co-localización de HvSS1 con el marcador de mitocondrias. Estos datos sugieren un papel distinto aunque parcialmente solapante de las cuatro Sacarosa Sintasas de cebada, descritas hasta la fecha. Las cinéticas de expresión de los genes que codifican los TFs más importantes implicados en la regulación génica durante el desarrollo del endospermo de cebada, se analizaron por RT-qPCR en ambos genotipos, demostrando que los TFs de la clase DOF aparecieron desregulados durante todo el proceso en Riso 1508 comparado con el cv. Bomi, aunque también se observaron diferencias significativas en algunos de los que codifican bZIPs. Estudios previos indicaban que el ortólogo de BLZ2 en maíz, O2, se regula post-traduccionalmente mediante un mecanismo de fosforilación/defosforilación reversible, y que la forma defosforilada es la fisiológicamente activa. En este trabajo se demostró que BLZ2 está sujeto a este tipo de regulación y que la proteín-fosfatasa HvPP2C2 está implicada en el proceso. La interacción de HvPP2C2 y BLZ2 tiene lugar en el núcleo celular únicamente en presencia de 100 μM ABA. En el mutante Riso 1508, BLZ2 se encuentra en un estado hiperfosforilado tanto durante la maduración como durante la germinación de la semilla, lo que dificultaría la unión de BLZ2 a las secuencias GLM en los promotores de los genes que codifican B-, C-,y ϒ- Hordeínas y CMe. Summary The seed is the main reproductive organ of spermatophyte plants allowing the spread of populations and ensuring their survival through its desiccation tolerance and because of their ability to germinate under optimum environmental conditions. Yield and economic value of cereal crops, that constitute the first world crop, depend largely on the efficiency with which they accumulate in the seed reserve substances: proteins, carbohydrates and lipids. The main carbohydrate accumulated in the barley seed is starch and the major protein fraction is that of prolamins (soluble in 70% ethanol); these proteins have a very low lysine content, an essential amino-acid for the diet of monogastric animals. In order to improve the nutritional value of the barley seed, different mutants have been obtained with a higher content of this amino-acid. Riso 1508 is one lysine-rich mutant whose mutation (lys3a) segregates as a single Mendelian gene with pleiotropic effects, such as a drastic reduction of genes encoding the trypsin inhibitor CMe and the B-, C-and ϒ-hordeins, but has not altered the expression of the gene encoding the D-hordeins. This latter gene lacks in its promotor the GLM motif (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), that is recognised by bZIP transcription factors In this work we have used the barley mutant Riso 1508 as a tool for better understanding gene regulation in seeds during the maturation and germination phases. To this aim, a transcriptomic analysis was performed comparing wild and mutant genotypes during seed maturation. Besides confirming variations in the expression of genes encoding reserve proteins, this analysis indicated that some genes related with carbohydrate metabolism were also affected. It was therefore decided to characterize the multigene family of sucrose synthases (SUSy) in barley. Two new genes were annotated, HvSs3 and HvSs4, and its expression was compared with that of genes HvSs1 and HvSs2, previously described in our laboratory. The expression of the four genes in different tissues and in response to abiotic stresses was analyzed by RTqPCR. HvSs1 and HvSs2 were preferentially expressed during the development of the endosperm, and the HvSs1 transcript was also abundant upon germination. HvSs1 was induced in leaves by anoxic conditions, HvSs3 by water stress, and both genes were induced by cold treatments. The subcellular localization of all four isoforms was not only cytoplasmic, but they could be found along the endoplasmic reticulum and at the inner side of the cell membrane; HvSS1, was also associated with the mitochondrial marker. These data suggest a distinct but partially overlapping roles for the barley sucrose synthases, described so far. The expression kinetics of the genes encoding the most important TFs involved in gene regulation during barley endosperm development was analyzed by RT-qPCR in both genotypes. These data show that the genes encoding DOF TFs were mis-regulated throughout the process in Riso 1508, although significant differences were also found among some of those encoding bZIPs. Previous studies indicated that the BLZ2 orthologue in maize, O2, was post-translationally regulated by reversible phosphorylation/dephosphorylation and that the dephosphorylated protein is the physiologically active form. In this work we demostrate that BLZ2 is under a similar regulation and that the proteinphosphatase HvPP2C2 is implicated in the process. The interaction between HvPP2C2 and BLZ2 takes place in the cell nucleus only in the presence of 100 μM ABA. In the Riso 1508 mutant, BLZ2 is found in a hyperphosphorylated state in the maturation phase and upon seed germination; because of this, the BLZ2 binding to the GLM promoter sequences of genes encoding B-, C- y ϒ- Hordeins and CMe would be decreased in the mutant.

Correlation between ileal digestibility of amino acids and chemical composition of soybean meals in broilers at 21 days of age

The correlations between chemical composition and coefficient of standardized ileal digestibility (CSID) of crude protein (CP) and amino acids (AA) were determined in 22 soybean meal (SBM) samples originated from USA (n = 8), Brazil (BRA; n = 7) and Argentina (ARG; n = 7) in 21-day old broilers. Birds were fed a commercial maize-SBM diet from 1 to 17 days of age followed by the experimental diets in which the SBM tested was the only source of protein (205 g CP/kg) for three days. Also, in vitro nitrogen (N) digestion study was conducted with these samples using the two-step enzymatic method. The coefficient of apparent ileal digestibility (CAID) of the SBM, independent of the origin, varied from 0.820 to 0.880 for CP, 0.850 to 0.905 for lysine (Lys), 0.859 to 0.907 for methionine (Met) and 0.664 to 0.750 for cysteine (Cys). The corresponding CSID values varied from 0.850 to 0.966 for CP, 0.891 to 0.940 for Lys, 0.931 to 0.970 for Met and 0.786 to 0.855 for Cys. The CSID of CP and Lys of the SBM were positively correlated with CP (r = 0.514; P menor que 0.05 and r = 0.370; P = 0.09, respectively), KOH solubility (KOH sol.) (r = 0.696; P menor que 0.001 and r = 0.619; P menor que 0.01, respectively), trypsin inhibitor activity (TIA) (r = 0.541; P menor que 0.01 and r = 0.416; P = 0.05, respectively) and reactive Lys (r = 0.563; P menor que 0.01 and r = 0.486; P menor que 0.05) values, but no relation was observed with neutral detergent fiber and oligosaccharide content. No relation between the CSID of CP determined in vivo and N digestibility determined in vitro was found. The CSID of most key AA were higher for the USA and the BRA meals than for the ARG meals. For Lys, the CSID was 0.921, 0.919 and 0.908 (P menor que 0.05) and for Cys 0.828, 0.833 and 0.800 (P menor que 0.01) for USA, BRA and ARG meals, respectively. It is concluded that under the conditions of this experiment, the CSID of CP and Lys increased with CP content, KOH sol., TIA and reactive Lys values of the SBM. The CSID of most limiting AA, including Lys and Cys, were higher for USA and BRA meals than for ARG meals.

Diagnosis and Management of Grain-Induced Asthma

Grain-induced asthma is a frequent occupational allergic disease mainly caused by inhalation of cereal flour or powder. The main professions affected are bakers, confectioners, pastry factory workers, millers, farmers, and cereal handlers. This disorder is usually due to an IgE-mediated allergic response to inhalation of cereal flour proteins. The major causative allergens of grain-related asthma are proteins derived from wheat, rye and barley flour, although baking additives, such as fungal α-amylase are also important. This review deals with the current diagnosis and treatment of grain-induced asthma, emphasizing the role of cereal allergens as molecular tools to enhance diagnosis and management of this disorder. Asthma-like symptoms caused by endotoxin exposure among grain workers are beyond the scope of this review. Progress is being made in the characterization of grain and bakery allergens, particularly cereal-derived allergens, as well as in the standardization of allergy tests. Salt-soluble proteins (albumins plus globulins), particularly members of the α-amylase/trypsin inhibitor family, thioredoxins, peroxidase, lipid transfer protein and other soluble enzymes show the strongest IgE reactivities in wheat flour. In addition, prolamins (not extractable by salt solutions) have also been claimed as potential allergens. However, the large variability of IgE-binding patterns of cereal proteins among patients with grain-induced asthma, together with the great differences in the concentrations of potential allergens observed in commercial cereal extracts used for diagnosis, highlight the necessity to standardize and improve the diagnostic tools. Removal from exposure to the offending agents is the cornerstone of the management of grain-induced asthma. The availability of purified allergens should be very helpful for a more refined diagnosis, and new immunomodulatory treatments, including allergen immunotherapy and biological drugs, should aid in the management of patients with this disorder.

Influence of origin of the beans on protein quality and nutritive value of commercial soybean meals.

Chemical composition and correlations between chemical analyses and protein quality of 454 batches of SBM of 3 different origins (USA, n=168; Brazil (BRA), n=139, and Argentine (ARG), n=147) were studied. Samples were collected during a 6-yr period. SBM from USA had more CP, sucrose and stachyose and less NDF (P<0.001) than SBM from ARG and BRA. CP content was negatively related (P<0.001) with sucrose for USA meals and with NDF for ARG and BRA meals. Also, P content was positively related (P<0.01) with CP content of the meals. PDI and KOH solubility were higher (P<0.001) for USA than for ARG or BRA SBM, values that were positively related (P<0.001) with trypsin inhibitor activity of the meals. In addition, USA meals had more lys, met+cys, thr, and trp than BRA and ARG meals (P < 0.001). Per unit of CP, lys content was negatively related (P<0.001) with CP content for USA, positively for BRA, and no relations was found for ARG meals. It is concluded that nutritive values and protein quality of the meals varied widely among soybean origins. Consequently, the origin of the beans should be considered in the evaluation of the nutritive value of commercial SBM for non-ruminant animals.

Influence of duration of storage on protein quality traits of soybean meals

Two experiments were conducted to determine the influence of duration of storage of soybean meal (SBM) on variables that define the quality of the protein fraction. Urease activity, protein dispersibility index (PDI), KOH protein solubility (KOHsol), and trypsin inhibitor activity were determined. In experiment 1, 8 samples of SBM, ranging in CP content from 55.4 to 56.5% DM, were collected from a US crushing plant at weekly intervals and analyzed at arrival to the laboratory and after 30, 60, 90, and 120 d of storage. In experiment 2, 7 samples of SBM, ranging in CP content from 49.0 to 55.0% DM, were collected from different Argentinean crushers and analyzed at arrival and after 24, 48, 80, and 136 wk of storage. In both experiments, samples were stored in hermetic glass containers in a laboratory room at 12 ± 2°C and a relative humidity of 70 ± 3%. Duration of storage did not affect urease activity or trypsin inhibitor activity values in either of the 2 experiments. However, PDI values decreased linearly with time of storage in both experiments (P menor que 0.001). Also, KOHsol decreased linearly (P menor que 0.05) with duration of storage in experiment 2 (long-term storage) but not in experiment 1(shorter term storage). Therefore, PDI values might not be adequate to compare protein quality of commercial SBM samples that have been stored for different periods of time. The KOHsol values are less affected by length of storage of the meals under current commercial practices.

N-Management using Nitrification inhibitor DMPP to Reduce Nitrogen Oxide Emissions in Mediterranean Winter Barley.

Increasing nitrogen (N) use efficiency during crop production is paramount both from an economic and environmental perspective. A proposed measure to achieve it is to split the addition of fertilizers with more than on application. For a winter crop under Mediterranean climatic conditions, the most common application pattern consists of a basal fertilization (October-November) an a top-dressing (February-March).

Soil moisture content determines the effect of the urease inhibitor NBPT on N2O emissions

Among the mitigation strategies to prevent nitrogen (N) losses from ureic fertilizers, urease inhibitors (UIs) have been demonstrated to promote high N use efficiency by reducing ammonia (NH3) volatilization. In the last few years, some field experiments have also shown its effectiveness in reducing nitrous oxide (N2O) losses from fertilized soils under conditions of low soil moisture. An incubation experiment was carried out with the aim of assessing the main biotic mechanisms behind N2O emissions once that the UIs N-(n-butyl) thiophosphoric triamid (NBPT) and phenil phosphorodiamidate (PPDA) were applied with Urea (U) under different soil moisture conditions (40, 60 and 80 % water-filled pore space, WFPS). In the same study we tried to analyze to what extent soil WFPS regulates the effect of these inhibitors on N2O emissions. The use of PPDA in our study allowed us to compare the effect of NBPT with that of another commercially available urease inhibitor, aiming to see if the results were inhibitor-specific or not. Based on the results from this experiment, a WFPS (i.e. 60 %) was chosen for a second study (i.e. mesocosm experiment) aiming to assess the efficiency of the UIs to indirectly affect N2O emissions through influencing the pool of soil mineral N. The N2O emissions at 40 % WFPS were almost negligible, being significantly lower from all fertilized treatments than that produced at 60 and 80 % WFPS. When compared to U alone, NBPT+U reduced the N2O emissions at 60 % WFPS but had no effect at 80 % WFPS. The application of PPDA significantly increased the emissions with respect to U at 80 % WFPS whereas no significant effect was found at 60 %. At 80 % WFPS, denitrification was the main source of N2O emissions for all treatments. In the mesocosm study, the application of NBPT+U was an effective strategy to reduce N2O emissions (75 % reduction compared to U alone), due to a lower soil ammonium (NH4 +) content induced by the inhibitor. These results suggest that adequate management of the UI NBPT could provide, under certain soil conditions, an opportunity for mitigation of N2O emissions from fertilized soils.