C1A Cysteine protease-cystatin interactions in leaf senescence

Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

Validation and aplication of Bioelectrical Impedance (BIA) method to determine body and carcass composition and nutrient retention of growing rabbits = Validación y aplicación de la técnica de la Impedancia Bioeléctrica (BIA) para determinar la composición corporal, de la canal y la retención de nutrientes de conejos en crecimiento

Los objetivos de esta tesis fueron 1) obtener y validar ecuaciones de predicción para determinar in vivo la composición corporal y de la canal de conejos en crecimiento de 25 a 77 días de vida utilizando la técnica de la Impedancia Bioeléctrica (BIA), y 2) evaluar su aplicación para determinar diferencias en la composición corporal y de la canal, así como la retención de nutrientes de animales alimentados con diferentes fuentes y niveles de grasa. El primer estudio se realizó para determinar y después validar, usando datos independientes, las ecuaciones de predicción obtenidas para determinar in vivo la composición corporal de los conejos en crecimiento. Se utilizaron 150 conejos a 5 edades distintas (25, 35, 49, 63 y 77 días de vida), con un rango de pesos entre 231 y 3138 g. Para determinar los valores de resistencia (Rs,) and reactancia (Xc,) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) de cada animal. En cada edad, los animales fueron molidos y congelados (-20 ºC) para su posterior análisis químico (MS, grasa, proteína, cenizas y EB). El contenido en grasa y energía de los animales se incrementó, mientras que los contenidos en proteína, cenizas y agua de los animales disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm y 10.8 ± 3.1 cm. Se realizó un análisis de regresión lineal múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) tuvieron un coeficiente de determinación de (R2) de 0.99, 0.99, 0.97, 0.98 y 0.99, y los errores medios de predicción relativos (EMPR) fueron: 2.79, 6.15, 24.3, 15.2 y 10.6%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.85 and 2.30%, respectivamente. Al predecir los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), se obtuvieron valores de 0.79, 0.83, 0.71 y 0.86 para R2, y 5.04, 18.9, 12.0 y 3.19% para EMPR. La reactancia estuvo negativamente correlacionada con el contenido en agua, cenizas y PB (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) y positivamente correlacionada con la grasa y la energía (r = 0.23 y r = 0.24; P < 0.01). Sin embargo, Rs estuvo positivamente correlacionada con el agua, las cenizas y la PB (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) y negativamente con la grasa y la energía (r = -0.36 y r = -0.35; P < 0.0001). Igualmente la edad estuvo negativamente correlacionada con el contenido en agua, cenizas y proteína (r = -0.79; r = -0.68 y r = -0.80; P < 0.0001) y positivamente con la grasa y la energía (r = 0.78 y r = 0.81; P < 0.0001). Se puede concluir que el método BIA es una técnica buena y no invasiva para estimar in vivo la composición corporal de conejos en crecimiento de 25 a 77 días de vida. El objetivo del segundo estudio fue determinar y validar con datos independientes las ecuaciones de predicción obtenidas para estimar in vivo la composición de la canal eviscerada mediante el uso de BIA en un grupo de conejos de 25 a 77 días, así como testar su aplicación para predecir la retención de nutrientes y calcular las eficacias de retención de la energía y del nitrógeno. Se utilizaron 75 conejos agrupados en 5 edades (25, 35, 49, 63 y 77 días de vida) con unos pesos que variaron entre 196 y 3260 g. Para determinar los valores de resistencia (Rs, ) y reactancia (Xc, ) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) del cada animal. En cada edad, los animales fueron aturdidos y desangrados. Su piel, vísceras y contenido digestivo fueron retirados, y la canal oreada fue pesada y molida para posteriores análisis (MS, grasa, PB, cenizas y EB). Los contenidos en energía y grasa aumentaron mientras que los de agua, cenizas y proteína disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. Se realizó un análisis de regresión linear múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Los coeficientes de determinación (R2) de las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) fueron: 0.99, 0.99, 0.95, 0.96 y 0.98, mientras que los errores medios de predicción relativos (EMPR) fueron: 4.20, 5.48, 21.9, 9.10 y 6.77%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.79 y 1.62%, respectivamente. Cuando se realizó la predicción de los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), los valores de R2 fueron 0.68, 0.76, 0.66 and 0.82, y los de RMPE: 3.22, 10.5, 5.82 and 2.54%, respectivamente. La reactancia estuvo directamente correlacionada con el contenido en grasa (r = 0.24, P < 0.05), mientras que la resistencia guardó una correlación positiva con los contenidos en agua, cenizas y proteína (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) y negativa con la grasa y la energía (r = -0.44 y r = -0.55; P < 0.001). Igualmente la edad estuvo negativamente correlacionada con los contenidos en agua, cenizas y PB (r = -0.94; r = -0.85 y r = -0.75; P < 0.0001) y positivamente con la grasa y la energía (r = 0.89 y r = 0.90; P < 0.0001). Se estudió la eficacia global de retención de la energía (ERE) y del nitrógeno (ERN) durante todo el periodo de cebo (35-63 d), Los valores de ERE fueron 20.4±7.29%, 21.0±4.18% and 20.8±2.79% en los periodos 35 a 49, 49 a 63 y 35 a 63 d, respectivamente. ERN fue 46.9±11.7%, 34.5±7.32% y 39.1±3.23% para los mismos periodos. La energía fue retenida en los tejidos para crecimiento con una eficiencia del 52.5% y la eficiencia de retención de la energía como proteína y grasa fue de 33.3 y 69.9% respectivamente. La eficiencia de utilización del nitrógeno para crecimiento fue cercana al 77%. Este trabajo muestra como el método BIA es técnica buena y no invasiva para determinar in vivo la composición de la canal y la retención de nutrientes en conejos en crecimiento de 25 a 77 días de vida. En el tercer estudio, se llevaron a cabo dos experimentos con el fin de investigar los efectos del nivel de inclusión y de la fuente de grasa, sobre los rendimientos productivos, la mortalidad, la retención de nutrientes y la composición corporal total y de la canal eviscerada de conejos en crecimiento de 34 a 63 d de vida. En el Exp. 1 se formularon 3 dietas con un diseño experimental factorial 3 x 2 con el tipo de grasa utilizada: Aceite de Soja (SBO), Lecitinas de Soja (SLO) y Manteca (L) y el nivel de inclusión (1.5 y 4%) como factores principales. El Exp. 2 también fue diseñado con una estructura factorial 3 x 2, pero usando SBO, Aceite de Pescado (FO) y Aceite de Palmiste como fuentes de grasa, incluidas a los mismos niveles que en el Exp. 1. En ambos experimentos 180 animales fueron alojados en jaulas individuales (n=30) y 600 en jaulas colectivas en grupos de 5 animales (n=20). Los animales alimentados con un 4% de grasa añadida tuvieron unos consumos diarios y unos índices de conversión más bajos que aquellos alimentados con las dietas con un 1.5% de grasa. En los animales alojados en colectivo del Exp. 1, el consumo fue un 4.8% más alto en los que consumieron las dietas que contenían manteca que en los animales alimentados con las dietas SBO (P = 0.036). La inclusión de manteca tendió a reducir la mortalidad (P = 0.067) en torno al 60% y al 25% con respecto a las dietas con SBO y SLO, respectivamente. La mortalidad aumentó con el nivel máximo de inclusión de SLO (14% vs. 1%, P < 0.01), sin observarse un efecto negativo sobre la mortalidad con el nivel más alto de inclusión de las demás fuentes de grasa utilizadas. En los animales alojados colectivo del Exp. 2 se encontró una disminución del consumo (11%), peso vivo a 63 d (4.8%) y de la ganancia diaria de peso (7.8%) con la inclusión de aceite de pescado con respecto a otras dietas (P < 0.01). Los dos últimos parámetros se vieron especialmente más reducidos cuando en las dietas se incluyó el nivel más alto de FO (5.6 y 9.5%, respectivamente, (P < 0.01)). Los animales alojados individualmente mostraron unos resultados productivos muy similares. La inclusión de aceite pescado tendió (P = 0.078) a aumentar la mortalidad (13.2%) con respecto al aceite de palmiste (6.45%), siendo intermedia para las dietas que contenían SBO (8.10%). La fuente o el nivel de grasa no afectaron la composición corporal total o de la canal eviscerada de los animales. Un incremento en el nivel de grasa dio lugar a una disminución de la ingesta de nitrógeno digestible (DNi) (1.83 vs. 1.92 g/d; P = 0.068 en Exp. 1 y 1.79 vs. 1.95 g/d; P = 0.014 en Exp. 2). Debido a que el nitrógeno retenido (NR) en la canal fue similar para ambos niveles (0.68 g/d (Exp. 1) y 0.71 g/d (Exp. 2)), la eficacia total de retención del nitrógeno (ERN) aumentó con el nivel máximo de inclusión de grasa, pero de forma significativa únicamente en el Exp. 1 (34.9 vs. 37.8%; P < 0.0001), mientras que en el Exp. 2 se encontró una tendencia (36.2 vs. 38.0% en Exp. 2; P < 0.064). Como consecuencia, la excreción de nitrógeno en heces fue menor en los animales alimentados con el nivel más alto de grasa (0.782 vs. 0.868 g/d; P = 0.0001 en Exp. 1, y 0.745 vs. 0.865 g/d; P < 0.0001 en Exp.2) al igual que el nitrógeno excretado en orina (0.702 vs. 0.822 g/d; P < 0.0001 en Exp. 1 y 0.694 vs. 0.7999 g/d; P = 0.014 en Exp.2). Aunque no hubo diferencias en la eficacia total de retención de la energía (ERE), la energía excretada en heces disminuyó al aumentar el nivel de inclusión de grasa (142 vs. 156 Kcal/d; P = 0.0004 en Exp. 1 y 144 vs. 154 g/d; P = 0.050 en Exp. 2). Sin embargo, la energía excretada como orina y en forma de calor fue mayor en el los animales del Exp. 1 alimentados con el nivel más alto de grasa (216 vs. 204 Kcal/d; P < 0.017). Se puede concluir que la manteca y el aceite de palmiste pueden ser considerados como fuentes alternativas al aceite de soja debido a la reducción de la mortalidad, sin efectos negativos sobre los rendimientos productivos o la retención de nutrientes. La inclusión de aceite de pescado empeoró los rendimientos productivos y la mortalidad durante el periodo de crecimiento. Un aumento en el nivel de grasa mejoró el índice de conversión y la eficacia total de retención de nitrógeno. ABSTRACT The aim of this Thesis is: 1) to obtain and validate prediction equations to determine in vivo whole body and carcass composition using the Bioelectrical Impedance (BIA) method in growing rabbits from 25 to 77 days of age, and 2) to study its application to determine differences on whole body and carcass chemical composition, and nutrient retention of animals fed different fat levels and sources. The first study was conducted to determine and later validate, by using independent data, the prediction equations obtained to assess in vivo the whole body composition of growing rabbits. One hundred and fifty rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) and weighing from 231 to 3138 g were used. A four terminal body composition analyser was used to obtain resistance (Rs, ) and reactance (Xc, ) values (Model BIA-101, RJL Systems, Detroit, MI USA). The distance between internal electrodes (D, cm), body length (L, cm) and live BW of each animal were also registered. At each selected age, animals were slaughtered, ground and frozen (-20 ºC) for later chemical analyses (DM, fat, CP, ash and GE). Fat and energy body content increased with the age, while protein, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm and 10.8 ± 3.1 cm. A multiple linear regression analysis was used to determine the prediction equations, using BW, L and Z data as independent variables. Equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content had, respectively, coefficient of determination (R2) values of 0.99, 0.99, 0.97, 0.98 and 0.99, and the relative mean prediction error (RMPE) was: 2.79, 6.15, 24.3, 15.2 and 10.6%, respectively. When water was expressed as percentage, the R2 and RMPE were 0.85 and 2.30%, respectively. When prediction of the content of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) was done, values of 0.79, 0.83, 0.71 and 0.86 for R2, and 5.04, 18.9, 12.0 and 3.19% for RMPE, respectively, were obtained. Reactance was negatively correlated with water, ash and CP content (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) and positively correlated with fat and GE (r = 0.23 and r = 0.24; P < 0.01). Otherwise, resistance was positively correlated with water, ash and CP (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) and negatively correlated with fat and energy (r = -0.36 and r = -0.35; P < 0.0001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.79; r = -0.68 and r = -0.80; P < 0.0001) and positively correlated with fat and energy (r = 0.78 and r = 0.81; P < 0.0001). It could be concluded that BIA is a non-invasive good method to estimate in vivo whole body composition of growing rabbits from 25 to 77 days of age. The aim of the second study was to determine and validate with independent data, the prediction equations obtained to estimate in vivo carcass composition of growing rabbits by using the results of carcass chemical composition and BIA values in a group of rabbits from 25 to 77 days. Also its potential application to predict nutrient retention and overall energy and nitrogen retention efficiencies was analysed. Seventy five rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) with weights ranging from 196 to 3260 g were used. A four terminal body composition analyser (Model BIA-101, RJL Systems, Detroit, MI USA) was used to obtain resistance (Rs, ) and reactance (Xc, ) values. The distance between internal electrodes (D, cm), body length (L, cm) and live weight (BW, g) were also registered. At each selected age, all the animals were stunned and bled. The skin, organs and digestive content were removed, and the chilled carcass were weighed and processed for chemical analyses (DM, fat, CP, ash and GE). Energy and fat increased with the age, while CP, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. A multiple linear regression analysis was done to determine the equations, using BW, L and Z data as parameters. Coefficient of determination (R2) of the equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content were: 0.99, 0.99, 0.95, 0.96 and 0.98, and relative mean prediction error (RMPE) were: 4.20, 5.48, 21.9, 9.10 and 6.77%, respectively. When water content was expressed as percentage, the R2 and RMPE were 0.79 and 1.62%, respectively. When prediction of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) content was done, R2 values were 0.68, 0.76, 0.66 and 0.82, and RMPE: 3.22, 10.5, 5.82 and 2.54%, respectively. Reactance was positively correlated with fat content (r = 0.24, P < 0.05) while resistance was positively correlated with water, ash and protein carcass content (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) and negatively correlated with fat and energy (r = -0.44 and r = -0.55; P < 0.001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.97, r = -0.95 and r = -0.89, P < 0.0001) and positively correlated with fat and GE (r = 0.95 and r = 0.97; P < 0.0001). In the whole growing period (35-63 d), overall energy retention efficiency (ERE) and nitrogen retention efficiency (NRE) were studied. The ERE values were 20.4±7.29%, 21.0±4.18% and 20.8±2.79%, from 35 to 49, 49 to 63 and from 35 to 63 d, respectively. NRE was 46.9±11.7%, 34.5±7.32% and 39.1±3.23% for the same periods. Energy was retained in body tissues for growth with an efficiency of approximately 52.5% and efficiency of the energy for protein and fat retention was 33.3 and 69.9%, respectively. Efficiency of utilization of nitrogen for growth was near to 77%. This work shows that BIA it’s a non-invasive and good method to estimate in vivo carcass composition and nutrient retention of growing rabbits from 25 to 77 days of age. In the third study, two experiments were conducted to investigate the effect of the fat addition and source, on performance, mortality, nutrient retention, and the whole body and carcass chemical composition of growing rabbits from 34 to 63 d. In Exp. 1 three diets were arranged in a 3 x 2 factorial structure with the source of fat: Soybean oil (SBO), Soya Lecithin Oil (SLO) and Lard (L) and the dietary fat inclusion level (1.5 and 4%) as the main factors. Exp. 2 had also arranged as a 3 x 2 factorial design, but using SBO, Fish Oil (FO) and Palmkernel Oil (PKO) as fat sources, and included at the same levels than in Exp. 1. In both experiments 180 animals were allocated in individual cages (n=30) and 600 in collectives cages, in groups of 5 animals (n=20). Animals fed with 4% dietary fat level showed lower DFI and FCR than those fed diets with 1.5%. In collective housing of Exp. 1, DFI was a 4.8% higher in animals fed with diets containing lard than SBO (P = 0.036), being intermediate for diet with SLO. Inclusion of lard also tended to reduce mortality (P = 0.067) around 60% and 25% with respect SBO and SLO diets, respectively. Mortality increased with the greatest level of soya lecithin (14% vs. 1%, P < 0.01). In Exp. 2 a decrease of DFI (11%), BW at 63 d (4.8%) and DWG (7.8%) were observed with the inclusion of fish oil with respect the other two diets (P < 0.01). These last two traits impaired with the highest level of fish oil (5.6 and 9.5%, respectively, (P < 0.01)). Animals housed individually showed similar performance results. The inclusion of fish oil also tended to increase (P = 0.078) mortality (13.2%) with respect palmkernel oil (6.45%), being mortality of SBO intermediate (8.10%). Fat source and level did not affect the whole body or carcass chemical composition. An increase of the fat sources addition led to a decrease of the digestible nitrogen intake (DNi) (1.83 vs. 1.92 g/d; P = 0.068 in Exp. 1 and 1.79 vs. 1.95 g/d; P = 0.014 in Exp. 2). As the nitrogen retained (NR) in the carcass was similar for both fat levels (0.68 g/d (Exp. 1) and 0.71 g/d (Exp. 2)), the overall efficiency of N retention (NRE) increased with the highest level of fat, but only reached significant level in Exp. 1 (34.9 vs. 37.8%; P < 0.0001), while in Exp. 2 a tendency was found (36.2 vs. 38.0% in Exp. 2; P < 0.064). Consequently, nitrogen excretion in faeces was lower in animals fed with the highest level of fat (0.782 vs. 0.868 g/d; P = 0.0001 in Exp. 1, and 0.745 vs. 0.865 g/d; P < 0.0001 in Exp.2). The same effect was observed with the nitrogen excreted as urine (0.702 vs. 0.822 g/d; P < 0.0001 in Exp. 1 and 0.694 vs. 0.7999 g/d; P = 0.014 in Exp.2). Although there were not differences in ERE, the energy excreted in faeces decreased as fat level increased (142 vs. 156 Kcal/d; P = 0.0004 in Exp. 1 and 144 vs. 154 g/d; P = 0.050 in Exp. 2). In Exp. 1 the energy excreted as urine and heat production was significantly higher when animals were fed with the highest level of dietary fat (216 vs. 204 Kcal/d; P < 0.017). It can be concluded that lard and palmkernel oil can be considered as alternative sources to soybean oil due to the reduction of the mortality, without negative effects on performances or nutrient retention. Inclusion of fish impaired animals´ productivity and mortality. An increase of the dietary fat level improved FCR and overall protein efficiency retention.

A global budget for fine root biomass, surface area, and nutrient contents

Global biogeochemical models have improved dramatically in the last decade in their representation of the biosphere. Although leaf area data are an important input to such models and are readily available globally, global root distributions for modeling water and nutrient uptake and carbon cycling have not been available. This analysis provides global distributions for fine root biomass, length, and surface area with depth in the soil, and global estimates of nutrient pools in fine roots. Calculated root surface area is almost always greater than leaf area, more than an order of magnitude so in grasslands. The average C:N:P ratio in living fine roots is 450:11:1, and global fine root carbon is more than 5% of all carbon contained in the atmosphere. Assuming conservatively that fine roots turn over once per year, they represent 33% of global annual net primary productivity.

Interactions between Senescence and Leaf Orientation Determine in Situ Patterns of Photosynthesis and Photoinhibition in Field-Grown Rice1

Photosynthesis and photoinhibition in field-grown rice (Oryza sativa L.) were examined in relation to leaf age and orientation. Two varieties (IR72 and IR65598-112-2 [BSI206]) were grown in the field in the Philippines during the dry season under highly irrigated, well-fertilized conditions. Flag leaves were examined 60 and 100 d after transplanting. Because of the upright nature of 60-d-old rice leaves, patterns of photosynthesis were determined by solar movements: light falling on the exposed surface in the morning, a low incident angle of irradiance at midday, and light striking the opposite side of the leaf blade in the afternoon. There was an early morning burst of CO2 assimilation and high levels of saturation of photosystem II electron transfer as incident irradiance reached a maximum level. However, by midday the photochemical efficiency increased again almost to maximum. Leaves that were 100 d old possessed a more horizontal orientation and were found to suffer greater levels of photoinhibition than younger leaves, and this was accompanied by increases in the de-epoxidation state of the xanthophyll cycle. Older leaves had significantly lower chlorophyll content but only slightly diminished photosynthesis capacity.

Water Deficit and Spatial Pattern of Leaf Development. Variability in Responses Can Be Simulated Using a Simple Model of Leaf Development1

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695–703).

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

pH-Regulated Leaf Cell Expansion in Droughted Plants Is Abscisic Acid Dependent1

Elongation rates of barley (Hordeum vulgare L. cv Hanna) leaves decreased with decreasing soil water content, whereas the pH of xylem sap increased from 5.9 to 6.9 over 6 d as the soil dried. The reduction in leaf-elongation rate (LER) was correlated with the increase in sap pH. Artificial sap buffered to different pH values was fed via the subcrown internode to derooted seedlings. Although leaves elongated at in planta rates when fed artificial sap at a well-watered pH of 6.0, LER declined with increasing sap pH. This effect persisted in the light and in the dark. pH had no effect on the relative water content or the bulk abscisic acid (ABA) concentration of the growing zone of these leaves. LERs of the ABA-deficient mutant Az34 were uniformly high over the pH range tested, whereas those of its isogenic wild-type cultivar Steptoe were reduced as the artificial sap pH was increased from 6.0 to 7.0. However, supplying a well-watered concentration of ABA (3 × 10−8 m) in the artificial xylem sap restored the pH response of the Az34 mutant. The results suggest that increased xylem sap pH acts as a drought signal to reduce LER via an ABA-dependent mechanism.

Does Leaf Position within a Canopy Affect Acclimation of Photosynthesis to Elevated CO2?1 : Analysis of a Wheat Crop under Free-Air CO2 Enrichment

Previous studies of photosynthetic acclimation to elevated CO2 have focused on the most recently expanded, sunlit leaves in the canopy. We examined acclimation in a vertical profile of leaves through a canopy of wheat (Triticum aestivum L.). The crop was grown at an elevated CO2 partial pressure of 55 Pa within a replicated field experiment using free-air CO2 enrichment. Gas exchange was used to estimate in vivo carboxylation capacity and the maximum rate of ribulose-1,5-bisphosphate-limited photosynthesis. Net photosynthetic CO2 uptake was measured for leaves in situ within the canopy. Leaf contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), light-harvesting-complex (LHC) proteins, and total N were determined. Elevated CO2 did not affect carboxylation capacity in the most recently expanded leaves but led to a decrease in lower, shaded leaves during grain development. Despite this acclimation, in situ photosynthetic CO2 uptake remained higher under elevated CO2. Acclimation at elevated CO2 was accompanied by decreases in both Rubisco and total leaf N contents and an increase in LHC content. Elevated CO2 led to a larger increase in LHC/Rubisco in lower canopy leaves than in the uppermost leaf. Acclimation of leaf photosynthesis to elevated CO2 therefore depended on both vertical position within the canopy and the developmental stage.

Temporal Regulation of Somatic Embryogenesis by Adjusting Cellular Polyamine Content in Eggplant1

Four critical stages of embryogenesis, including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic program, and development and maturation of somatic embryos during somatic embryogenesis from leaf discs of eggplant (Solanum melongena L.), were identified by scanning electron microscopy. Temporal changes in arginine decarboxylase (ADC) activity and polyamines (PAs) during critical stages of embryogenesis revealed that high levels of PAs (especially putrescine [PUT]), due to higher ADC activity in discs from the apical region (with high embryogenic capacity) than from the basal region of the leaf (with poor embryogenic capacity), were correlated with differential embryogenesis response. Kinetic studies of the up- and down-regulation of embryogenesis revealed that PUT and difluoromethylarginine pretreatments were most effective before the onset of embryogenesis. Basal discs pretreated with PUT for 4 to 7 d showed improved embryogenesis that was comparable to apical discs. PA content at various critical steps in embryogenesis from basal discs were found to be comparable to that of apical discs following adjustments of cellular PA content by PUT. In contrast, pretreatment of apical discs with difluoromethylarginine for 3 d significantly reduced ADC activity, cellular PA content, and embryogenesis to levels that were comparable to basal discs. Discs from the basal region of leaves treated with PUT for 3 d during the identified stages of embryogenesis improved their embryogenic potential.

Influence of light on DNA content of Helianthus annuus Linnaeus.

Mean nuclear 2C DNA content (C equaling haploid DNA per nucleus) of the first leaf of the sunflower, Helianthus annuus L., is influenced by the quality and the quantity of light. Seedlings of two inbred lines, RHA 299 and RHA 271 were germinated and grown in controlled environmental conditions. Lighting was adjusted to provide different combinations of photon flux densities and red to far red (R:FR) ratios. At R:FR = 5.8 and photon flux densities of 170 mumol.m-2.s-1, 200 mumol.m-2.s-1, and 230 mumol.m-2.s-1, DNA content remained high and relatively constant (x = 6.97 pg for RHA 271 and x = 7.32 pg for RHA 299). When the photon flux density range (R:FR = 5.8) was elevated to 350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1, mean DNA content was reduced to 6.23 pg (RHA 271) and 6.46 pg (RHA 299). At R:FR = 1.5, mean DNA content was consistently high (7.2-7.9 pg) only at the lowest photon flux density of 170 mumol.m-2.s-1. Significant decreases in DNA content (< or = 12%) were observed at photon flux densities of 200 mumol.m-2.s-1 and 230 mumol.m-2.s-1. At the higher photon flux densities (350 mumol.m-2.s-1, 410 mumol.m-2.s-1, and 470 mumol.m-2.s-1) and R:RF = 1.5, the plants had extremely low DNA contents (mean x = 3.36 pg for RHA 271 and 3.41 pg for RHA 299) and high between-plant variance. The instability of DNA content, particularly for plants grown under light that is far red rich, suggests that phytochromes may be involved in regulating DNA content of the sunflower.

Solute losses from various shoot parts of field-grown wheat by leakage in the rain

Leakage from field-grown wheat was investigated during two seasons differing considerably in their rainfall patterns. For all solutes analyzed, these losses were low from non-senescing plant parts, increased after the onset of senescence and became maximal in fully senesced (dry, brown) organs. The cumulative losses of potassium by leakage in the rain were 65% of the content at anthesis for the flag leaf and 95% for the third leaf from the top, while these relative values were lower for magnesium (50 to 80%) and calcium (around 55%) and extremely low for sodium (<10%). The differences between potassium and sodium may be due to a different compartmentation on the tissue level or on the subcellular level. It became evident that for certain nutrients (e.g. potassium or magnesium) leakage in the rain may represent a major loss from senescing leaves and can be a relevant flux in maturing wheat.

(Tab. XIII.1) TOC content, carbonate content, carbonate mineralogy, percentage of total carbonate, aragonite, low magnesium calcium, and high magnesium calcium, and grain-size contents from samples from the Golfe d'Arguin and from the Western Sahara

Modern carbonate sedimentation takes place on the northern Mauritanian shelf (20°N), where typical tropical components (e.g. hermatypic reefs, calcareous green algae) are absent. Such deposits are reminiscent of extratropical sediment in the geological record. The tropical open shelf of Mauritania is influenced by large siliciclastic dust input and upwelling, highly fertilizing the ocean, as well as strongly limiting the light penetration. In this context, temperature does not appear to be the steering factor of carbonate production. This thesis describes the depositional system of the Golfe d'Arguin off Mauritania and focuses on environmental conditions that control the depositional pattern, in particular carbonate production. The description of this modern analogue provides a tool for paleoenvironmental interpretation of ancient counterparts. The Golfe d'Arguin is a broad shallow shelf comprising extensive shoals (<10 m water depth; i.e. the Banc d'Arguin) on the inner shelf where waters warm up. The sediments collected in water depths between 4 and 600 m are characterized by mixed carbonate and siliciclastic (dust) deposits. They vary from clean coarse-grained, almost pure carbonate loose sediments to siliciclastic-dominated fine-grained sediments. The carbonate content and sediment grain size show a north-south decreasing pattern through the Golfe d'Arguin and are controlled by the hydraulic regime influenced by wind-driven surface currents, swell, and tidal currents. The carbonate grain association is heterozoan. Components include abundant molluscs, foraminifers, and worm tubes, as well as barnacles and echinoderms, elements that are also abundant in extratropical sediments. The spatial distribution of the sedimentary facies of the Golfe d'Arguin does not display a depth zonation but rather a mosaic (i.e. patchy distribution). The depth and climatic signatures of the different sedimentary facies are determined by taxonomic and ecological investigations of the carbonate-secreting biota (molluscs and foraminifers). While certain planktonic foraminifers and molluscs represent upwelling elements, other components (e.g. mollusc and benthic foraminifer taxa) demonstrate the tropical origin of the sediment. The nutrient-rich (and thus also low light-penetration) conditions are reflected in the fact that symbiotic and photosynthetic carbonate-producing organisms (e.g. hermatypic corals) are absent. The Mauritanian deposits represent an environment that is rare in the modern world but might have been more common in the geological past when global temperatures were higher. Taxonomic and ecological studies allow for distinguishing carbonate sediments formed under either tropical high-nutrient or extratropical conditions, thus improving paleoclimate reconstruction.

(Table 1) Carbon content and petroleum potential at DSDP Hole 73-520

At the end of the Leg 73 cruise, at Site 520, a thick upper Miocene facies of laminated diatomite was discovered. It was hypothesized that the sediment was derived from nutrient-rich waters in a young Cenozoic anoxic basin in the open Atlantic Ocean near the Mid-Atlantic Ridge.

Layer description, carbon and nitrogen content and stable isotope record of bulk sedimentary organic matter from Laguna Potrok Aike

An investigation of stable isotope (d13C TOC and d15N TN) and elemental parameters (TOC, TN contents and TOC/TN ratios) of bulk organic matter (<200 µm) from sediment cores recovered from the Patagonian lake Laguna Potrok Aike (Argentina) in the framework of the ICDP deep drilling project PASADO provided insights into past changes in lake primary productivity and environmental conditions in South Patagonia throughout the last Glacial-Interglacial transition. Stratigraphically constrained cluster analyses of all proxy parameters suggest four main phases. From ca 26,100 to 17,300 cal. years BP, lacustrine phytoplankton was presumably the predominant organic matter source in an aquatic environment with low primary productivity rates. At around 17,300 cal. years BP, abrupt and distinct shifts of isotopic and elemental values indicate that the lacustrine system underwent a rapid reorganization. Lake primary productivity (phytoplankton and aquatic macrophytes) shows higher levels albeit with large variations during most of the deglaciation until 13,000 cal. years BP. The main causes for this development can be seen in improved growing conditions for primary producers because of deglacial warming in combination with expedient availability of nutrients and likely calm wind conditions. After 13,000 cal. years BP, decreased d13C TOC values, TOC, TN contents and TOC/TN ratios indicate that the lake approached a new state with reduced primary productivity probably induced by unfavourable growing conditions for primary producers like strengthened winds and reduced nutrient availability. The steady increase in d15N TN values presumably suggests limitation of nitrate supply for growth of primary producers resulting from a nutrient shortage after the preceding phase with high productivity. Nitrate limitation and consequent decreased lacustrine primary productivity continued into the early Holocene (10,970-8400 cal. years BP) as reflected by isotopic and elemental values.

(Table A1) Calcium carbonate content of boreholes Kirchrode I and II

The technical details of drilling and coring at the Kirchrode I and II sites are presented. At these sites, a sequence of claystones and marlstones from an Albian shelf basin was recovered. Constraints on the ages of the sediments in the two boreholes are provided by the occurrence of the inoceramid bivalve Actinoceramus sulcatus, the first appearance of which is used to define the Middle/Upper Albian boundary and by observed facies changes that can be correlated to the established lithostratigraphy. The cores from the two boreholes provide a rather complete, 285-m-long sequence of the Upper Albian, with a 155.5-m-long overlap. Analysis of the tectonic structures showed considerable shortening in the Middle and Lower Albian part of the sequence due to normal faulting. Of the Upper Albian, only the lowermost part is affected by faults. The increase in sedimentation rates of terrigenous detritus and of marine biogenic carbonate, which occurs in the basal part of the C. auritus Subzone, is interpreted to reflect a regional change to a more humid climate and regional tectonic movements (uplift of the Rhenish Bohemian massif, subsidence of the Lower Saxony basin intensified locally by halokinetic movements). The further increase in marine productivity in the latest Albian may be related to upwelling of more nutrient-rich deep water along submarine relief in this shelf sea. Identification of Milankovitch cyclicity documented by the fluctuating CaCO3 contents of the sediments is used (i) to constrain the minimum time represented by the Upper Albian deposits, and (ii) to determine the duration of the sea level cycles (Cycle V: >=1.6 Ma, Cycle VI: >=2 Ma), and (iii) to establish the duration of the Late Albian ammonite subzones (e.g. Callihoplites auritus Subzone: 2.1 Ma). Average sedimentation rates determined from the identified 100-ka eccentricity cycles show a stepwise increase in sedimentation rates from 1-2 cm/1000 a in the Lower Albian dark claystones to 7-13 cm/1000 a in the late Late Albian. In addition to the general deepening trend through the Late Albian, two, nearly completely documented 3rd-order sea-level cycles in the Upper Albian of Kirchrode I were recognised, plus another one, cut short by faulting, at the base of the Upper Albian (documented in Kirchrode II). These global sea-level cycles were identified on the basis (a) of the sequence of the abundance maxima of selected benthos and plankton groups, (b) of trends in the fluctuations of the CaCO3 content, and (c) of the abundance of glauconite. The transgression periods in this Upper Albian deep shelf-basin are characterised by intensified circulation. This intensified circulation is found to have affected first the surface-near waters, resulting e.g. in an increase in the abundance of immigrant plankton and nekton species from the Tethys. At a later stage the deep water was affected, supporting then an increased population of suspension-feeding benthos, and causing condensation and erosion in the sediment at the sea floor.