Modelling sugar beet water use in Spain

Crop production has a great contribution to water use and abstraction. Sugar beet is an important crop in irrigated land in Spain and covers 70.000 Ha. Crop and resources management are key factors for a sustainable agriculture. The aim of this work is to mode the sugar beet crop growth and water consumption in order to quantify crop water use and virtual water content in different growing conditions.

Identification of the method to quantify soluble fibre and the effect of the source of fibre on the ileal and faecal digestibility of soluble and insoluble fibre in rabbits = Identificación del método para cuantificar la fibra soluble y el efecto de la fuente de fibra en la digestibilidad ileal y fecal de la fibra soluble e insoluble en conejos

La presente tesis constituye un avance en el estudio de los métodos para cuantificar la fibra soluble y los efectos de las fracciones de fibra y las fuentes de fibra sobre la digestión de las diferentes fracciones de fibra (soluble e insoluble) en el conejo. Hay un efecto positivo de la fibra soluble sobre la salud intestinal de los conejos y, por ende, una reducción de la mortalidad en animales destetados. Pese a esto, no está claro si estos efectos se deben específicamente a la fracción soluble. Por lo que los objetivos generales de esta tesis fueron: 1) comparar diferentes metodologías químicas e in vitro para cuantificar la fibra soluble y estudiar las posibles interferencias en la cuantificación de la fibra soluble por las mucinas, y viceversa, 2) determinar los efectos de la fibra, el lugar de fermentación, el método para valorar la fibra soluble e insoluble, y la corrección de la fibra soluble por el contenido intestinal de mucinas sobre la digestibilidad de las distintas fracciones de la fibra y 3) evaluar los efectos individuales de las fracciones soluble e insoluble de la fibra de pulpa de remolacha y de manzana, sobre la digestibilidad de la fibra soluble e insoluble y los parámetros digestivos. Para ello se llevaron a cabo 4 estudios. En el primer estudio se compararon diferentes metodologías químicas e in vitro para valorar la fibra soluble de diferentes alimentos y se estudió la posible interferencia en la determinación de la fibra soluble y mucinas. Para ello se utilizaron seis ingredientes (pulpa de remolacha, pectinas de pulpa de remolacha, pulpa de remolacha lavada, paja de cereal, cascarilla de girasol y lignocelulosa) y siete piensos de conejos con diferentes niveles de fibra soluble. En un primer experimento se analizó la fibra dietética total (FDT), la fibra dietética insoluble (FDI), la fibra dietética soluble (FDS), la fibra neutro detergente corregida por cenizas y proteínas (aFNDmo-pb), y la digestibilidad in vitro 2 pasos pepsina/pancreatina (residuo corregido por cenizas y proteína, ivMSi2) de los ingredientes y piensos. Además la fibra soluble se calculó mediante la diferencia entre FDT-FDI (FDSFDI), FDT- ivMSi2 (FDSivMSi2), y FDT - aFNDmo-pb (FDSaFNDmo-pb). Cuando la fibra soluble se determinó directamente como FDS o se calculó como FDT-FDI no se observaron diferencias (109 g/kg MS, en promedio). Sin embargo, cuando la fibra soluble se calculó como FDT - aFNDmo-pb su valor fue un 40% menor (153 g/kg MS. P < 0,05), mientras que la FDSFDI (124 g/kg MS) no fue diferente a ninguna de las otras metodologías. La correlación entre los tres métodos fue elevada (r > 0,96. P < 0,001. n = 13), pero disminuyó o incluso desapareció cuando la pulpa o las pectinas de la remolacha fueron excluidas del análisis. En un segundo experimento, se comparó el método ivDMi2 usando crisoles (método de referencia) con una modificación del mismo usando bolsas ANKOM digeridas individualmente o en colectivo para simplificar la determinación de la FDSivMSi2. La FDSivMSi2 no difirió entre los métodos comparados. En un tercer experimento, se analizó la posible interferencia entre la determinación de la fibra soluble y las mucinas intestinales. Se observó un contenido de FDT y de mucinas elevado en las muestras de pectinas de remolacha (994 y 709 g/kg MS), así como en el moco intestinal de conejo (571 y 739 g/kg MS) cuando se aplicó el método de mucinas por precipitación con etanol. Sin embargo, después de aplicar una pectinasa en el material precipitado, la cantidad de mucinas recuperadas en las muestras de pectinas de remolacha fue cercana a cero, mientras que en el moco intestinal fue similar a los resultados previos al uso de la enzima. Con los resultados de este ensayo se estimaron los carbohidratos de mucinas retenidos en los contenidos digestivos y se propuso una corrección para la determinación de la digestibilidad de la FDT y fibra soluble. En conclusión, la contaminación de las mucinas de la digesta con fibra soluble se soluciona usando pectinasas. El segundo estudio se centró en estudiar: 1) el efecto del tipo de fibra, 2) el sitio de fermentación, 3) el método para cuantificar fibra y 4) la corrección por mucinas sobre la digestibilidad de la fibra. Para ello se formularon tres piensos con diferentes niveles de fibra soluble (FDT-aFNDmo-pb). Un pienso bajo en fibra soluble (LSF. 85 g/kg DM), un pienso medio en fibra soluble (MSF. 102 g/kg DM), y un pienso alto en fibra soluble (HSF. 145 g/kg DM). Estos piensos se obtuvieron reemplazando un 50% del heno del alfalfa en el pienso MSF por una mezcla de pulpa de manzana y remolacha (HSF) o por una mezcla de cascarilla de avena y proteína de soja (LSF). Se utilizaron 30 conejas canuladas para determinar la digestibilidad ileal y fecal. La digestibilidad cecal se calculó mediante diferencia entre la digestibilidad fecal e ileal. La fibra insoluble se determinó como aFNDmo-pb, IDF, e ivMSi2, mientras que la fibra soluble se calculó como FDSFDI, FDSaFNDmo-pb, y FDSivMSi2. La digestibilidad de la FDT y la fibra soluble se corrigieron por las mucinas. La concentración de mucinas en la digesta ileal y fecal, aumento desde el grupo LSF hasta el grupo con el pienso HSF (P < 0,01). La corrección por mucinas aumentó las digestibilidades de la FDT y la fibra soluble a nivel ileal, mientras que a nivel cecal las redujo. (P < 0.01). El coeficiente de digestibilidad ileal de FDT aumentó desde el grupo LSF al grupo HSF (0,12 vs. 0,281. P < 0,01), sin diferencias en el coeficiente de digestibilidad cecal (0,264), por lo que la tendencia a nivel fecal entre los grupos se mantuvo. El coeficiente de digestibilidad ileal de la fibra insoluble aumento desde el grupo con el pienso LSF al grupo con el pienso HSF (0,113 vs. 0,210. P < 0,01), sin diferencias a nivel cecal (0,139) y sin efecto del método usado, resultando en una digestibilidad elevada a nivel fecal, con tendencias similares a las observadas a nivel ileal. El coeficiente de digestibilidad de la FND fue elevada en comparación con la FDI o la ivMSi2 (P > 0.01). El coeficiente de la digestibilidad ileal de la fibra soluble fue mayor en el grupo LSF respecto al grupo LSF (0,436 vs. 0,145. P < 0,01) y el método no afectó a esta determinación. El coeficiente de la digestibilidad cecal de la fibra soluble se redujo desde el grupo LSF hasta el grupo HSF (0,721 vs. 0,492. P < 0,05). El valor más bajo de digestibilidad cecal y fecal de fibra soluble fue medido con el método FDSaFNDmo-pb (P < 0,01). Se observó una alta correlación entre las digestibilidades de la fibra soluble determinada como FDSFDI, FDSaFNDmo-pb, y FDSivMSi2, por lo tanto la información proporcionada por una u otra metodología fueron similares. Sin embargo, cuando se compararon con efectos fisiológicos (producción de mucinas y peso del ciego y pH del ciego de un trabajo previo), la FDSaFNDmo-pb globalmente mostró estar mejor correlacionado con estos parámetros fisiológicos. En conclusión, la corrección por mucinas es necesaria para determinar la digestibilidad ileal de la FDT y fibra soluble, mientras que la elección de uno u otro método es menos relevante. La inclusión de pulpa de manzana y remolacha incrementa la cantidad de FDT que desaparece antes de llegar al ciego. En el tercer estudio se estudió el efecto de la fracción fibrosa soluble e insoluble de la pulpa de remolacha y el método de cuantificación de la fibra soluble e insoluble sobre la digestibilidad de la fibra y algunos parámetros digestivos. Para ello se formularon cuatro piensos con niveles similares de fibra insoluble (315g aFNDmo-pb/kg MS) y proteína (167 g/kg MS). El pienso control contuvo el nivel más bajo de fibra soluble (30,3 g/kg, con cascarilla de girasol y paja como fuente de fibra). Un segundo pienso se obtuvo mediante la sustitución de 60 g de almidón/kg del pienso control por pectinas de remolacha (82,9 g fibra soluble/kg MS). Los otras dos piensos resultaron de la sustitución parcial de las fuentes de fibra del pienso control por la fracción insoluble de la pulpa de remolacha y la pulpa de remolacha entera (42.2 y 82.3 g fibra soluble/kg MS, respectivamente). Cincuenta y seis conejos en cebo (14/pienso), de 2,4  0.21 kg de peso, fueron usados para determinar la digestibilidad ileal y fecal de la FDT, FDI, aFNDmo-pb, FDSFDI, y FDSaFNDmo-pb. La concentración de mucinas en el íleon y heces se utilizaron para corregir la digestibilidad de la FDT y fibra soluble. También se midió el peso de diferentes segmentos del tracto digestivo y el pH del contenido digestivo. Los conejos alimentados con el pienso de fibra insoluble de pulpa de remolacha mostraron los consumos más bajos con respecto a los demás grupos (124 vs. 139 g/d, respectivamente. P < 0,05). El flujo de mucinas ileales fue más alto (P < 0.05) en el grupo alimentado con el pienso de pectinas de remolacha (9,0 g/d en promedio) que los del grupo control (4,79 g/d), mostrando los otros dos grupos valores intermedios, sin detectarse diferencias a nivel fecal. La digestibilidad ileal de la FDT (corregida por mucinas) y la fibra insoluble no se vieron afectadas por el tipo de pienso. El método usado para determinar la fibra insoluble afectó su digestibilidad ileal (0,123 para FDI vs. 0,108 para aFNDmo-pb. P < 0.01). De todas formas, los métodos no afectaron al cálculo de la fibra fermentada antes del ciego (4,9 g/d en promedio). Los conejos alimentados con el pienso de pulpa de remolacha y con el pienso con la fracción insoluble de la pulpa de remolacha mostraron las digestibilidades fecales más altas de la fibra insoluble (0,266 en promedio vs. 0,106 del grupo control), mientras que en los animales del pienso con pectinas esta digestibilidad fue un 47% mayor respecto al pienso control (P < 0,001). La digestibilidad fecal de la fibra insoluble fue un 20% más alta cuando se usó la FND en lugar de FDI para determinarla (P < 0.001). Esto hizo variar la cantidad de fibra insoluble fermentada a lo largo del tracto digestivo (9,5 ó 7,5 g/d cuando fue calculada como FDI o aFNDmo-pb, respectivamente. P < 0,001). Las digestibilidades ileales de la fibra soluble fueron positivas cuando los análisis de fibra soluble de los contenidos ileales fueron corregidos por mucinas, (P < 0,001) excepto para la digestibilidad ileal de la FDSIDF del grupo control. Una vez corregidas por mucinas, los conejos alimentados con los piensos que contuvieron la fracción soluble de la pulpa de remolacha (pienso de pectina y pulpa de remolacha) mostraron una mayor digestibilidad ileal de la fibra soluble, respecto al grupo control (0,483 vs. -0,010. P = 0.002), mientras que el grupo del pienso de fibra insoluble de pulpa de remolacha mostró un valor intermedio (0,274). La digestibilidad total de la fibra soluble fue similar entre todos los grupos (0.93). Los conejos alimentados con pulpa de remolacha y su fracción insoluble mostraron los pesos relativos más altos del estómago respecto a los del pienso control y de pectinas (11 y 56 % respectivamente; P < 0,05). Por otra parte, el peso relativo del ciego aumentó en los animales que consumieron tanto la fracción soluble como insoluble de la pulpa de remolacha, siendo un 16% más pesados (P < 0,001) que el grupo control. El pH del contenido cecal fue más bajo en los animales del grupo de pulpa de remolacha que en los del grupo control (5,64 vs. 6,03; P < 0,001), mientras que los del grupo de pectinas y de fibra insoluble de pulpa de remolacha mostraron valores intermedios. En conclusión, el efecto positivo de la pulpa de remolacha en el flujo de mucinas a nivel ileal se debe a la fracción soluble e insoluble de la pulpa de remolacha. La mitad de la fibra soluble de la pulpa de remolacha desaparece antes de llegar al ciego, independientemente si esta proviene de pectinas puras o de la pulpa de remolacha. El pH cecal esta mejor correlacionado con la cantidad de FDT que desaparece antes del ciego más que con la que se degrada en el ciego. En el último estudio se estudiaron los efectos de la fibra soluble e insoluble de la pulpa de manzana sobre la digestibilidad de la fibra y algunos parámetros digestivos. Cuatro dietas fueron formuladas con niveles similares de fibra insoluble (aFNDmo-pb 32,4%) y proteína (18,6% ambos en base seca). El pienso control contuvo el nivel más bajo de fibra soluble (46 g de fibra soluble/kg, con cascarilla de girasol y paja de cereales como la fuentes de fibra). Un segundo pienso fue obtenido mediante la sustitución de 60 g de almidón/kg del pienso control por pectinas de manzana (105 g fibra soluble/kg). Los otros dos piensos se obtuvieron por la substitución de parte de las fuentes de fibra del pienso control por pulpa de manzana o pulpa de manzana despectinizada (93 y 71 g de fibra soluble/kg, respectivamente). La digestibilidad fecal fue determinada en 23 conejos/pienso con 1.68 ± 0.23 kg de peso vivo, los cuales fueron sacrificados a los 60 d edad para recolectar su contenido digestivo para determinar digestibilidad ileal y otros parámetros digestivos. La fibra soluble de manzana (pectinas y pulpa entera) estimuló el flujo ileal de mucinas (P = 0,002), pero no asi la pulpa despectinizada. La corrección por mucinas incrementó la digestibilidad de la FDT y la fibra soluble a nivel fecal, y especialmente a nivel ileal. Cerca de la mitad de la fibra soluble proveniente de los piensos con cualquiera de las fracciones de la pulpa de manzana fue degradada a nivel ileal, sin mostrar diferencias entre los grupos (46 y 86% en promedio a nivel ileal y fecal respectivamente). La inclusión de pulpa despectinizada de manzana mejoró la digestibilidad de la FND a nivel fecal (P < 0,05) pero no a nivel ileal. El contenido cecal de los conejos alimentados con la pulpa de manzana tuvieron el pH cecal más ácido que los del pienso control (5,55 vs. 5,95. P < 0,001), mientras que los animales con el pienso de pectinas de manzana y de pulpa de manzana despectinizada mostraron valores intermedios. En conclusión los efectos positivo de la pulpa de manzana en el flujo de mucinas se debió principalmente a la fracción soluble de la pulpa de manzana. La mitad de la fibra soluble fue degradada antes del ciego independientemente de si esta provino de las pectinas o de la pulpa de manzana. El pH cecal estuvo mejor correlacionado con la cantidad de FDT fermentada en todo el tracto digestivo y antes de llegar al ciego que con la que se degradó en el ciego. Al integrar los resultados de los estudio 2, 3 y 4 se concluyó que la corrección de mucinas de los contenidos digestivos al determinar FDT y fibra soluble es necesaria para ajustar los cálculos de su digestibilidad. Esta corrección es mucho más importante a nivel ileal y en dietas bajas en fibra soluble. Por otra parte, la FDT desapareció en proporciones importantes antes de llegar al ciego, especialmente en piensos que contienen pulpa de remolacha o de manzana o alguna fracción soluble o insoluble de las mismas y estas diferencias observadas entre los piensos a nivel ileal se correlacionaron mejor con el pH cecal, lo que indicaría que la FDT se solubilizó antes de llegar al ciego y una vez en esté fermentó. Estos resultados implican que determinar la fibra soluble como FDSaFNDmo-pb es la mejor opción y que en la determinación de la digestibilidad de la FDT y fibra soluble se debe considerar la corrección por mucinas especialmente a nivel ileal y en piensos bajos en fibra soluble. ABSTRACT The present thesis constitutes a step forward in advancing the knowledge of the methods to quantify soluble fibre and the effects of the fibre fractions and source of fibre on the site the digestion of different fractions of fibre (soluble and insoluble) in the rabbit. There is a positive effect of soluble fibre on rabbit digestive health and therefore on the reduction of mortality in weaning rabbits. Nevertheless, it is no so clear that the effects of soluble fibre on rabbits are due particularly to this fraction. This thesis aims: 1) to compare the quantification of soluble fibre in feeds using different chemical and in vitro approaches, and to study the potential interference between soluble fibre and mucin determinations, 2) to identify the effects of type of fibre, site of fermentation, method to quantify insoluble and soluble fibre, and correction of the intestinal soluble fibre content for intestinal mucin on the digestibility of fibre fractions and 3) to evaluate the individual effect of soluble and insoluble fibre from sugar beet pulp and apple pulp on ileal and faecal soluble and insoluble digestibility and digestive traits. These objectives were developed in four studies: The first study compared the quantification of soluble fibre in feeds using different chemical and in vitro approaches, and studied the potential interference between soluble fibre and mucin determinations. Six ingredients, sugar beet pulp (SBP), SBP pectins, insoluble SBP, wheat straw, sunflower hulls and lignocellulose, and seven rabbit diets, differing in soluble fibre content, were evaluated. In experiment 1, ingredients and diets were analysed for total dietary fibre (TDF), insoluble dietary fibre (IDF), soluble dietary fibre (SDF), aNDFom (corrected for protein, aNDFom-cp) and 2-step pepsin/pancreatin in vitro DM indigestibility (corrected for ash and protein, ivDMi2). Soluble fibre was estimated by difference using three procedures: TDF - IDF (SDFIDF), TDF - ivDMi2 (SDFivDMi2), and TDF - aNDFom-cp (SDFaNDFom-cp). Soluble fibre determined directly (SDF) or by difference, as SDFivDMi2 were not different (109 g/kg DM, on average). However, when it was calculated as SDFaNDFom-cp the value was 40% higher (153 g/kg DM, P < 0.05), whereas SDFIDF (124 g/kg DM) did not differ from any of the other methods. The correlation between the four methods was high (r ≥ 0.96. P ≤ 0.001. n = 13), but it decreased or even disappeared when SBP pectins and SBP were excluded and a lower and more narrow range of variation of soluble fibre was used. In experiment 2, the ivDMi2 using crucibles (reference method) were compared to those made using individual or collective ankom bags in order to simplify the determination of SDFivDMi2. The ivDMi2 was not different when using crucibles or individual or collective ankom bags. In experiment 3, the potential interference between soluble fibre and intestinal mucin determinations was studied using rabbit intestinal raw mucus, digesta and SBP pectins, lignocelluloses and a rabbit diet. An interference was observed between the determinations of soluble fibre and crude mucin, as the content of TDF and apparent crude mucin were high in SBP pectins (994 and 709 g/kg DM) and rabbit intestinal raw mucus (571 and 739 g/kg DM). After a pectinase treatment, the coefficient of apparent mucin recovery of SBP pectins was close to zero, whereas that of rabbit mucus was not modified. An estimation of the crude mucin carbohydrates retained in digesta TDF is proposed to correct TDF and soluble fibre digestibility. In conclusion, the values of soluble fibre depend on the methodology used. The contamination of crude mucin with soluble fibre is avoided using pectinase. The second study focused on the effect of type of fibre, site of fermentation, method for quantifying insoluble and soluble dietary fibre, and their correction for intestinal mucin on fibre digestibility. Three diets differing in soluble fibre were formulated (85 g/kg DM soluble fibre, in the low soluble fibre [LSF] diet; 102 g/kg DM in the medium soluble fibre [MSF] diet; and 145 g/kg DM in the high soluble fibre [HSF] diet). They were obtained by replacing half of the dehydrated alfalfa in the MSF diet with a mixture of beet and apple pulp (HSF diet) or with a mix of oat hulls and soybean protein (LSF diet). Thirty rabbits with ileal T-cannulas were used to determine total tract apparent digestibility (CTTAD) and ileal apparent digestibility (CIAD). Caecal digestibility was determined by difference between CTTAD and CIAD. Insoluble fibre was measured as aNDFom-cp, IDF, and ivDMi2, whereas soluble fibre was calculated as SDFaNDFom-cp, SDFIDF, SDFivDMi2. The intestinal mucin content was used to correct the TDF and soluble fibre digestibility. Ileal and faecal concentration of mucin increased from the LSF to the HSF diet group (P < 0.01). Once corrected for intestinal mucin, The CTTAD and CIAD of TDF and soluble fibre increased whereas caecal digestibility decreased (P < 0.01). The CIAD of TDF increased from the LSF to the HSF diet group (0.12 vs. 0.281. P < 0.01), with no difference in the caecal digestibility (0.264), resulting in a higher CTTAD from the LSF to the HSF diet group (P < 0.01). The CIAD of insoluble fibre increased from the LSF to the HSF diet group (0.113 vs. 0.21. P < 0.01), with no difference in the caecal digestibility (0.139) and no effect of fibre method, resulting in a higher CTTAD for rabbits fed the HSF diet compared with the MSF and LSF diets groups (P < 0.01). The CTTAD of aNDFom-cp was higher compared with IDF or ivDMi2 (P < 0.01). The CIAD of soluble fibre was higher for the HSF than for the LSF diet group (0.436 vs. 0.145. P < 0.01) and fibre method did not affect it. Caecal soluble fibre digestibility decreased from the LSF to the HSF diet group (0.721 vs. 0.492. P < 0.05). The lowest caecal and faecal soluble fibre digestibility was measured using SDFaNDFom-cp (P < 0.01). There was a high correlation among the digestibilities of soluble fibre measured as SDFaNDFom-cp, SDFIDF, and SDFivDMi2. Therefore, these methodologies provide similar information. However, the method that seems to be globally better related to the physiological traits (ileal flow of mucins, and relative weight of the caecum and caecal pH from previous work) was the SDFaNDFom-cp. In conclusion, a correction for intestinal mucin is necessary for ileal TDF and soluble fibre digestibility whereas the selection of the fibre method has a minor relevance. The inclusion of sugar beet and apple pulp increased the amount of TDF fermented in the small intestine. The third study examined the effect of fibre fractions of sugar beet pulp (SBP) and the method for quantifying soluble and insoluble fibre on soluble and insoluble fibre digestibility and digestive traits. Four diets were formulated with similar level of insoluble fibre (aNDFom-cp: 315 g/kg DM) and protein (167 g/kg DM). Control diet contained the lowest level of soluble fibre (30.3 g/kg DM, including sunflower hulls and straw as sole sources of fibre). A second diet was obtained by replacing 60 g starch/kg of control diet with SBP pectins (82.9 g soluble fibre/kg DM). Two more diets were obtained by replacing part of the fibrous sources of the control diet with either insoluble SBP fibre or SBP (42.2 and 82.3 g soluble fibre/kg DM, respectively). Fifty six (14/diet) rabbits weighing 2.40  0.213 kg were used to determine faecal and ileal digestibility of total dietary fibre (TDF), insoluble dietary fibre (IDF), neutral detergent fibre corrected for ash and CP (aNDFom-cp) and soluble fibre estimated as SDFaNDFom-cp and SDFIDF. Faecal and ileal mucin content was used to correct TDF and soluble fibre digestibility. It was also recorded weight of digestive segments and digesta pH. Rabbits fed insoluble SBP showed the lowest feed intake with respect to the other 3 diets (124 vs. 139 g/d, respectively. P < 0.05). Ileal mucin flow was higher (P < 0.05) in animals fed pectin and SBP diets (9.0 g/d, as average) than those fed control diet (4.79 g/d), showing InsSBP group an intermediate value. No differences on mucin content were detected at faecal level. There was no diet effect on the CIAD of TDF (corrected for mucin) and insoluble fibre. Fibre methodology influenced the CIAD of insoluble fibre (0.123 for IDF vs. 0.108 for aNDFom-cp. P < 0.01). Anyway, the amount of insoluble fibre fermented before the caecum did not differ between both methods (4.9 g/d, on average). Rabbits fed insoluble SBP and SBP diets showed the highest CTTAD of insoluble fibre (0.266 on average vs. 0.106 for control group), whereas those fed pectin diet had an intermediate value (0.106. P < 0.001). The CTTAD of insoluble fibre measured with IDF was higher than that measured with aNDFom-cp (by 20%. P < 0.001). It led that the amount of insoluble fibre fermented along the digestive tract were different (9.5 or 7.5 g/d when calculated as IDF or aNDFom-cp, respectively; P < 0.001). When the CIAD of soluble fibre was corrected for mucin they became positive (P < 0.001) except for control group measured as SDFIDF. Once corrected for mucin content, rabbits fed soluble fibre from SBP (pectin and SBP groups) showed higher CIAD of soluble fibre than control group (0.483 vs. -0.019. respectively), whereas the value for insoluble SBP group was intermediate 0.274. The CTTAD of soluble fibre (mucin corrected) was similar among diets 0.93. Rabbits fed with SBP and insoluble SBP diets showed higher total digestive tract and stomach relative weight than those fed pectin and control diets (by 11 and 56 %. respectively, P < 0.05). The caecal relative weight did not differ in rabbits fed pectin, insoluble SBP, and SBP diets (62 g/kg BW, as average) and they were on average 16% higher (P < 0.001) than in control group. Caecal content of rabbits fed SBP diet was more acid than those fed control diet (5.64 vs. 6.03. P < 0.001), whereas those from pectin and insoluble SBP diets showed intermediate values. In conclusion, the positive effect of SBP fibre on ileal mucin flow was due to both its soluble and insoluble fibre fraction. Half of the soluble SBP fibre was degraded before the caecum independently it came from pectin or SBP. The caecal pH correlated better with the ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. The last study examined the effect of soluble and insoluble fibre of apple pulp on fibre digestibility and digestive traits. Four diets were formulated with similar level of insoluble fibre (aNDFom-cp: 324 g/kg DM) and protein (18.6 g/kg DM). Control diet contained the lowest level of soluble fibre (46 g soluble fibre/kg DM, including oat hulls and straw as sole sources of fibre). A second diet was obtained by replacing 60 g starch/kg of control diet with apple pectins (105 g soluble fibre/kg DM). Two more diets were obtained by substituting part of the fibrous sources of the control diet by either apple pulp or depectinized apple pulp (93 and 71 g soluble fibre/kg, respectively). The CTTAD was determined in 23 rabbits/diet weighing 1.68  0.23 kg BW, and 23 rabbits/diet were slaughtered at 60 d of age to collect ileal digesta to determine CIAD and record other digestive traits. Soluble fibre from apple stimulated ileal flow of mucin (P = 0.002), but depectinized apple pulp did not. The correction for mucin increased the digestibility of crude protein, total dietary fibre, and soluble fibre at faecal, but especially at ileal level, depending in this case on the diet. Around half of the soluble fibre in diets containing any fibre fraction from apple was degraded at ileal level, with no differences among these diets (0.46 vs. 0.066 for control group, P=0.046). Faecal soluble fibre digestibility was 0.86 on average for all groups). Inclusion of the apple insoluble fibre improved NDF digestibility at faecal (0.222 vs. 0.069. P < 0.05) but not at ileal level. Caecal content of rabbits fed apple pulp diet was more acid than those fed control diet (5.55 vs. 5.95. P < 0.001), whereas those from pectin and depectinised apple pulp diets showed intermediate values. In conclusion, the positive effect of apple fibre on ileal mucin flow was mainly due to its soluble fibre fraction. Half of the soluble apple fibre was degraded before the caecum independently it came from pectin or apple pulp. The caecal pH correlated better with the total and ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. The results obtained in the studies 2, 3 and 4 were considered together. These results showed that the mucin correction is necessary when the TDF and soluble fibre digestibility is determined, and it correction is more important at ileal level and in diets with low level of soluble fibre. On another hand, incrementing the soluble fibre using sugar beet and apple pulp increased the amount of TDF disappear before the caecum. Moreover, the caecal pH correlated better with the ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. This suggests that an ileal fibre solubilisation may occur rather than ileal fermentation. Therefore the implications of this work were that: the estimation of soluble fibre as SDFaNDFom-cp is an adequate method considering its correlation with the physiological effects; and the TDF and soluble fibre digestibility must be corrected with intestinal mucins, especially when the ileal digestibility is determined.

Ca-amendement and tillage: medium term synergies for improving key soil properties of acid soils. Soil and tillage research, 134: 195-206

Ca-amendments are routinely applied to improve acid soils, whilst no-tillage (NT) has been widely recommended in soils where traditional tillage (TT) has led to losses of organic matter. However, the potential interactions between the two treatments are only partially known. Our study was conducted on an annual forage crop agrosystem with a degraded Palexerult soil located in SW Spain, in order to assess if the combination of NT plus a Ca-amendment provides additional benefits to those of their separate use. To this end we analysed the effects of four different combinations of tillage and Ca-amendment on selected key soil properties, focusing on their relationships. The experimental design was a split-plot with four replicates. The main factor was tillage (NT versus TT) and the second factor was the application or not of a Ca-amendment, consisting of a mixture of sugar foam (SF) and red gypsum (RG). Soil samples were collected from 3 soil layers down to 50 cm after four years of treatment (2009). The use of the Ca-amendment improved pH and Al-toxicity down to 25 cm and increased exchangeable Ca2+ down to 50 cm, even under NT due to the combined effect of SF and RG. Both NT and the Ca-amendment had a beneficial effect on total organic carbon (TOC), especially on particulate organic carbon (POC), in the 0–5 cm layer, with the highest contents observed when both practices were combined. Unlike NT, the Ca-amendment failed to improve soil aggregation in spite of the carbon supplied. This carbon was not protected within the stable aggregates in the medium term, making it more susceptible to mineralization. We suggest that the fraction of Al extracted by oxalate from solid phase (AlOxa-Cu-K) and the glomalin-related soil proteins (GRSPs) are involved in the accumulation of carbon within water stable aggregates, probably through the formation of non-toxic stable Al-OM compounds, including those formed with GRSPs. NT alone decreased AlK in the 0–5 cm soil layer, possibly by increasing POC, TOC and GRSPs, which were observed to play a role in reducing Al toxicity. From our findings, the combination of NT and Ca-amendment appears to be the best management practice to improve chemical and physical characteristics of acid soils degraded by tillage.

Genetic control of abscisic acid biosynthesis in maize

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C15 intermediate xanthoxin and C25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9′-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pPfruB-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pPfruB-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to ≈1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Heterozygous reeler mice (HRM) haploinsufficient for reelin express ≈50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD67)-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD67 down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD67 knockout mice (HG67M). These mice exhibited a down-regulation of GAD67 mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose

Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.

Identification of thermophilic species by the amino acid compositions deduced from their genomes

The global amino acid compositions as deduced from the complete genomic sequences of six thermophilic archaea, two thermophilic bacteria, 17 mesophilic bacteria and two eukaryotic species were analysed by hierarchical clustering and principal components analysis. Both methods showed an influence of several factors on amino acid composition. Although GC content has a dominant effect, thermophilic species can be identified by their global amino acid compositions alone. This study presents a careful statistical analysis of factors that affect amino acid composition and also yielded specific features of the average amino acid composition of thermophilic species. Moreover, we introduce the first example of a ‘compositional tree’ of species that takes into account not only homologous proteins, but also proteins unique to particular species. We expect this simple yet novel approach to be a useful additional tool for the study of phylogeny at the genome level.

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping1

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves1

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m−2. Mesophyll conductance (μ) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on Et saturated at Et = 30 μmol active sites m−2 and μ = 11 mm s−1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a μ of only 6 to 8 mm s−1. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm−1 s−1. Our data show that the saturation of the relationship between Et and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.

A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood

The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH2-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.

Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities in Vicia sativa Seedlings1

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mm solution of MetJA stimulated ω-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min−1 mg−1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid ω-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.