Expression profiling and sequence diversity of novel DREB genes from common bean (Phaseolus vulgaris L.) and their association with drought-related traits

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance

Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris (L.) leaves

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713--1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) alpha-amylase inhibitor transgene

The nutritive value of transgenic peas expressing an a-amylase inhibitor (alpha-Ail) was evaluated with broiler chickens. The effects of feeding transgenic peas on the development of visceral organs associated with digestion and nutrient absorption were also examined. The chemical composition of the conventional and the transgenic peas used in this study were similar. In the two feeding trials, that were conducted normal and transgenic peas were incorporated into a maize-soybean diet at concentrations up to 500 g kg(-1). The diets were balanced to contain similar levels of apparent metabolisable energy (AME) and amino acids. In the first trial, the birds were fed the diets from 3 to 17days post-hatching and with levels of transgenic peas at 250 g kg(-1) or greater there was a significant reduction in body weight but an increase in feed intake resulting in deceased feed conversion efficiency. In the second trial, in which the birds were fed diets containing 300 g kg(-1) transgenic peas until 40 days of age, growth performance was significantly reduced. It was also demonstrated that the ileal starch digestibility coefficient (0.80 vs 0.42) was significantly reduced in the birds fed transgenic peas. Determination of AME and ileal digestibility of amino acids in 5-week-old broilers demonstrated a significant reduction in AME (12.12 vs 5.08 MJ kg(-1) DM) in the birds fed the transgenic peas. The AME value recorded for transgenic peas reflected the lower starch digestibility of this line. Real digestion of protein and amino acids was unaffected by treatment. Expression of a-Ail in peas did not appear to affect bird health or the utilisation of dietary protein. However, the significant reduction in ileal digestion of starch in transgenic peas does reduce the utility of this feedstuff in monogastric diets where efficient energy utilisation is required. (c) 2006 Society of Chemical Industry.

Concentración de cationes (Ca2+, Mg2+, Fe2+3+, K+) en semillas de frijol negro (Phaseolus vulgaris L.) usando espectroscopia de absorción atómica

Se investiga la concentración de cationes (Ca,Fe,K,Mg),en el frijol negro,(Phaseolus vulgaris L. var. Tineco), usando un analizador de absorción atómica. Para tal efecto se procede a secar la muestra y luego se digiere en un medio acido. Se preparan las soluciones standard correspondiente a los cationes que se quiere investigar y luego se comparan estas concentraciones con las concentraciones de los iones en la muestra. El cálculo de la concentración (desconocida), de los cationes en la muestra, se hace por medio de simple aritmética. En este trabajo se reporta la cantidad en p. p. m. de los elementos arriba mencionados, presente en un gramo de la muestra original. Los resultados obtenidos fueron: Fe = 337.5 p. p. m. ; Ca = 150 p. p. m. ; Mg = 150 p. p. m. y K = 4X104p. p. m.

Caracterización, evaluación preliminar y adaptabilidad de cuatro poblaciones de frijol común (Phaseolus vulgaris L.), evaluadas en cinco localidades de Ciudad Darío, Matagalpa; Postrera, 2013

En el estudio realizado en el municipio de Ciudad Dar í o, Matagalpa, é poca de postrera 2013, se sometieron cuatro variedades de frijol com ú n ( Phaseolus vulgaris L.), a los diferentes ambientes de las localidades El Moj ó n, El Rinc ó n, El Pital, Dulce Nombre de Jes ú s y la Picota donde se evaluaron dos variedades locales (Guanise ñ o, Rojo Maravilla), una aclimatada a la zona (Rojo seda), y una variedad mejorada (INTA sequia). Para el ensayo se establecieron cuatro parcelas de 10 m de largo por 6 m de ancho, en donde la parcela ú til tuvo una dimensi ó n 6 m de largo y 3 m de ancho, cabe mencionar que cada parcela estaba constituida de 5 surcos, de las que se evaluaron 5 plantas por surco para un total de 25 plantas por cada parcela, adem á s cada finca se consider ó una r é plica. Los objetivos planteados fueron 1) Caracterizar y evaluar preliminarmente mediante variables cuantitativas y cualitativas cuatro cultivares de frijol (dos criollas, una acriollada y una mejorada), en cinco localidades de Ciudad Dar í o, Matagalpa, en la é poca de postrera 2013 y 2) Determinar la adaptabilidad de cuatro variedades de frijol com ú n (dos criollas, una acriollada y una mejorada), en base a los rendimientos obtenidos por cada variedad en estudio. En el siguiente estudio se realiz ó un an á lisis de adaptabilidad el cual se llev ó a cabo por medio del an á lisis de regresi ó n lineal, adem á s se evalu ó el comportamiento de los caracteres cualitativos y cuantitativos para determinar la variabilidad gen é tica existentes en las diferentes variedades. En donde se demuestra que en los caracteres cualitativos no existi ó variabilidad dado que estos caracteres son gobernados por uno o pocos genes, en cambio se pudo observar que en los caracteres cuantitativos existe alta heterogeneidad entre los mismos dado que estos caracteres son influenciados por el ambiente, adem á s se conoci ó cu á l de las cuatro variedades en estudio present ó mejor adaptabilidad en las diferentes localidades, tambi é n se informa sobre la variedad por la que el productor debe optar para la siembra del cultivo antes mencionado

Diagnóstico de suelos en cultivos de café (Coffea arabica L.), maíz (Zea mays L.) y frijol (Phaseolus vulgaris L.), comunidad de Santa Julia, El Crucero, Managua

Uno de los parámetros de mayor importancia en la agricultura es el estado nutricional del suelo, así como el manejo de parte de los agricultores. En 2014, a través del programa para el Desarrollo Participativo Integral Rural (DEPARTIR)se efectuó un muestreo de suelos a profundidad de 25 cm en 34 lotes productivos de 17 unidades de producción, dedicadas a la producción de café (Coffea arabica), maíz ( Zea mays L.) y frijol (Phaseolusvulgaris L.) en la comunidad Santa Julia, El Crucero, Managua. Se analizaron los factores que condicionan el uso del suelo, a través de análisis de correlación, ANDEVA y análisis de correspondencia en N, P,K, Mg, Ca, MO, CO y pH. El N total,y altitudfueron correlacionadas. El 95% de las áreas presentaron 10.5 hectáreas, con predominancia decultivo de café. Lafertilización químicaes unapráctica común en las unidades productivasy cultivos estudiados. Los muestreos efectuados en las parcelas de maíz fueron homogéneos a lo interno y externo de las unidades productivas; no así en el cultivo de café. Las áreas de siembra, destino de la producción y la aplicación de fertilizantes químicos, presentaron una alta relación.

Adaptabilidad y estabilidad de cuatro variedades de frijol (Phaseolus vulgaris L.) en seis localidades de San Dionisio, Matagalpa, en dos ciclos agrícolas, postrera 2013 y primera 2014

El cultivo del Frijol ( Phaseolus vulgaris L.) es uno de los principales rubro s que producen los agricultores del Municipio de San Dionisio , Matagalpa, haciendo uso de diferentes variedades criollas y acriolladas persistentes en la zona. El objetivo de ésta investigación fue estudiar la variabilidad fenotípica y la adaptabilidad de t res variedades criollas ( Guaniseño, Rojo seda y Rack sequía) y una variedad acriollada (INTA Sequía) durante d os ciclos agrícolas ( Postrera 2013 y Primera 2014 ) . Para esto , se establecieron las parcelas experimentales en una finca por localidad (6), l as dimensiones de las parcelas fueron 5 m de ancho por 10 m de largo y la parcela útil consistió de 3 m de ancho y 6 m de longitud. Para la variabilidad fenotípica se analizaron variables cuantitativas de crecimiento y desarrollo de las plantas a través de medidas de tendencias estadísticas y para el análisis de adaptabilidad se analizó la estabilidad del rendimiento de las variedades en las dife rentes localidades por medio de Análisis de Varianza y de Regresión Lineal . Entre los resul tados obtenidos se enc ontró que todas las variedades criollas y acriolladas evaluadas presentaron mayor variabilidad fenotípica en el ciclo agrícola postrera 2013. Las variables emergencia, vaina s por planta, plantas cosechadas y rendimiento por parcela mostraron los coeficientes de variación más altos con valores que oscilan entre 17.96 y 56.01 , principalmente la variable rendimiento p or parcela que fue la que mostró los valores más altos. En cuan to a la adaptabilidad de las variedades , el modelo de regresión lineal no resultó apropiado para analizar los datos obtenidos en ambos ciclos , sin embargo , se pudo tener una idea de la estabilidad de las variedades a través de la Regresión de la Desviación en donde se encontró que la varied ad Rack Sequia fue la que mostró menor coeficiente de Desviación (8.22) lo que demuestra que sus rendimientos fueron los más estables a nivel de fincas y ciclos agrícolas por lo tanto, fue la mejor adaptada a los diferentes ambientes.

Inoculaci?n y establecimiento endof?tico de cepas de los hongos entomopat?genos beauveria bassiana, Metarhizium anisopliae y lecanicillium lecanii en plantas de fr?jol (Phaseolus vulgaris) [recurso electr?nico]

Los hongos end?fitos han recibido una creciente atenci?n en los a?os recientes, ya que han demostrado tener un potencial econ?mico en la agronom?a, debido a su posible uso como agentes de control biol?gico. El establecimiento endof?tico de hongos entomopat?genos en cultivos de inter?s, ofrece un campo amplio y prometedor para comprender las relaciones entre plantas y hongos y los mecanismos de defensa que estos ?ltimos pueden inducir en las plantas. El presente estudio busc? inocular y evaluar la colonizaci?n endof?tica de tres especies de hongos entomopat?genos en plantas de fr?jol (Phaseolus vulgaris). Para ello, se emplearon dos cepas de cada especie, una comercial y la otra de la colecci?n de entomopat?genos del Centro Internacional de Agricultura Tropical; cada cepa constituy? un tratamiento individual. A partir de la siembra y desarrollo de las cepas, se generaron in?culos de 108 conidios/mL que fueron inoculados en las plantas de fr?jol Diacol, variedad Calima. Se encontr? que Beauveria bassiana, Metarhizium anisopliae y Lecanicillium lecanii colonizaron individualmente las hojas de fr?jol. Los porcentajes de colonizaci?n promedio para las cepas de la colecci?n del CIAT fueron de 16% (?= 18,84) para B. bassiana, 13% (?= 4,96) en M. anisopliae y 4% (?= 4,64) para L. lecanii. Con respecto a las cepas comerciales, estos valores fueron 4% (?= 4,64) para B. bassiana; 26% (?= 32) para M. anisopliae y 44% (?= 13,02) para L. lecanii. Todas las cepas de hongos inoculadas pudieron ser recuperadas de tejido vegetal, lo que sugiere su posible uso como agentes de control biol?gico al menos para el cultivo del fr?jol.

Caracterização fisio-molecular de cultivares de feijão (Phaseolus vulgaris L.) em resposta à deficiência hídrica

The identification of genotypes for drought tolerance has a great importance in breeding programs. The aim of this study was to characterize genotypes of beans in response to drought tolerance in different reproductive stages through physiologic, agronomic and molecular analysis. The experiment was conducted in greenhouse, using a randomized block design with four replicates; 10 cultivars: ANFC 9, ANFP 110, BRS Esplendor, BRSMG Realce, IPR Siriri, IPR Tangará, IPR Tuiuiu, IPR Uirapuru, IAC Imperador and IAC Milênio under two conditions of irrigation: plants irrigated during their entire life cycle, and plants under irrigation suppression in the reproductive stage (R7) until 16% of field capacity, when the irrigation was restored. In the last four days of stress, the gas exchanges were analyzed, and in the last day of stress was analyzed the percentage of closed stomata in the abaxial surface of the leaves, collected in different times of the day (9h, 12h, 15h and 18h). Additionally, plant samples were collected for the following analysis: fresh and dry mass of leaves, stems and legumes, and proline content in leaves and roots. The plants were harvested at the physiological maturity and the yield components and grain yield were determined. In addition, in order to identify polymorphisms in the sequences of promoters and genes related to drought, seven pairs of primers were tested on the group of genotypes. The drought susceptibility indexes (ISS) ranged from 0.65 to 1.10 in the group of genotypes, which the lowest values observed were for IAC Imperador (0.65) and BRS Esplendor (0.87), indicating the ability of these two genotypes to maintain grain yield under water stress condition. All genotypes showed reduction in yield components under water stress. IAC Imperador (43.4%) and BRS Esplendor (60.6%) had the lowest reductions in productivity and kept about 50% of the stomata closed during all the different times evaluated at last day of irrigation suppression. IAC Imperador showed greater water use efficiency and CO2 assimilation rate under drought stress. IPR Tuiuiú, IPR Tangará and IAC Imperador had the highest proline concentrations in the roots. Under water stress condition, there was a strong positive correlation (0.696) between the percentage of stomata closed with the number of grains per plant (0.696) and the fresh mass of leaves (0.731), the maximum percentage of stomata closed 73.71% in water stress. The accumulation of proline in the root was the character that most contributed to the divergence between the genotypes under water deficit, but not always the genotypes that have accumulated more proline were the most tolerant. The polymorphisms in DNA of coding and promoting sequences of transcription factors studied in this experiment did not discriminate tolerant genotypes from the sensitive ones to water stress.

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Physiological and molecular analysis of the interaction between aluminium toxicity and drought stress in common bean (Phaseolus vulgaris)

Aluminium (Al) toxicity and drought are two major factors limiting common bean (Phaseolus vulgaris) production in the tropics. Short-term effects of Al toxicity and drought stress on root growth in acid, Al-toxic soil were studied, with special emphasis on Al-drought interaction in the root apex. Root elongation was inhibited by both Al and drought. Combined stresses resulted in a more severe inhibition of root elongation than either stress alone. This result was different from the alleviation of Al toxicity by osmotic stress (-0.60 MPa polyethylene glycol) in hydroponics. However, drought reduced the impact of Al on the root tip, as indicated by the reduction of Al-induced callose formation and MATE expression. Combined Al and drought stress enhanced up-regulation of ACCO expression and synthesis of zeatin riboside, reduced drought-enhanced abscisic acid (ABA) concentration, and expression of NCED involved in ABA biosynthesis and the transcription factors bZIP and MYB, thus affecting the regulation of ABA-dependent genes (SUS, PvLEA18, KS-DHN, and LTP) in root tips. The results provide circumstantial evidence that in soil, drought alleviates Al injury, but Al renders the root apex more drought-sensitive, particularly by impacting the gene regulatory network involved in ABA signal transduction and cross-talk with other phytohormones necessary for maintaining root growth under drought.

Alteration of cell-wall porosity is involved in osmotic stress-induced enhancement of aluminium resistance in common bean (Phaseolus vulgaris L.)

Aluminium (Al) toxicity and drought are the two major abiotic stress factors limiting common bean production in the tropics. Using hydroponics, the short-term effects of combined Al toxicity and drought stress on root growth and Al uptake into the root apex were investigated. In the presence of Al stress, PEG 6000 (polyethylene glycol)-induced osmotic (drought) stress led to the amelioration of Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1. PEG 6000 (>> PEG 1000) treatment greatly decreased Al accumulation in the 1 cm root apices even when the roots were physically separated from the PEG solution using dialysis membrane tubes. Upon removal of PEG from the treatment solution, the root tips recovered from osmotic stress and the Al accumulation capacity was quickly restored. The PEG-induced reduction of Al accumulation was not due to a lower phytotoxic Al concentration in the treatment solution, reduced negativity of the root apoplast, or to enhanced citrate exudation. Also cell-wall (CW) material isolated from PEG-treated roots showed a low Al-binding capacity which, however, was restored after destroying the physical structure of the CW. The comparison of the Al(3+), La(3+), Sr(2+), and Rb(+) binding capacity of the intact root tips and the isolated CW revealed the specificity of the PEG 6000 effect for Al. This could be due to the higher hydrated ionic radius of Al(3+) compared with other cations (Al(3+) >> La(3+) > Sr(2+) > Rb(+)). In conclusion, the results provide circumstantial evidence that the osmotic stress-inhibited Al accumulation in root apices and thus reduced Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1 is related to the alteration of CW porosity resulting from PEG 6000-induced dehydration of the root apoplast.

Determinación de análisis bromatológico proximal y calidad culinaria de Phaseolus vulgaris L. (frijol común) grano negro criollo cultivado en la zona occidental de El Salvador

Esta investigación apuesta a la utilización del Phaseolus vulgaris L. (frijol común) grano negro criollo como un recurso que aumente el acceso alimenticio de las familias salvadoreñas. Para ello se estudió la calidad culinaria y bromatológica de tres variedades de Phaseolus vulgaris L. (frijol común) grano negro criollo que respalde el fomento del consumo de esta leguminosa. Los análisis realizados comprende la calidad culinaria que consiste en la determinación del coeficiente de hidratación, tiempo de cocción, densidad del caldo y las dimensiones del grano; y el análisis bromatológico proximal que consiste en la determinación de cenizas, humedad, proteína cruda, fibra cruda, extracto étereo, extracto libre de nitrógeno y minerales de importancia tales como: hierro, calcio, magnesio y zinc. Las muestras son variedades de Phaseolus vulgaris L. (frijol común) grano negro criollo (Cuarentín, Tamazulapa y El Porvenir) cultivadas en la zona occidental de El Salvador. Los resultados se compararon con los valores de referencia de frijol grano negro seco presentados en las tablas de composición de alimentos del Instituto de Nutrición de Centroamérica y Panamá (INCAP), y se evaluaron estadísticamente a través del análisis de varianza de un factor, análisis de diferencia significativa honesta de Tukey y representación gráfica Biplot. La variedad de frijol común grano negro criollo con mejores resultados en cuanto a calidad culinaria fue la variedad Cuarentín, en cambio en calidad bromatológica y cuantificación de micronutrientes los mejores resultados fueron para las variedades Cuarentín y Tamazulapa; sin embargo, la variedad con mayor cantidad proteica fue la variedad El Porvenir.

Physical and chemical properties of starch and flour from different common bean (Phaseolus vulgaris L.) cultivar.

The physical, chemical and pasting properties of the flour and isolated starches from six different bean cultivars (Phaseolus vulgaris L.) were investigated in order to obtain information for application in new products. The protein and total starch contents of the bean flours ranged from 17.72 to 20.27% and from 39.68 to 43.78%, respectively. The bean starches had low amounts of proteins, lipids and ash and showed an amylose content ranging between 45.32 and 51.11% and absolute density values between 1.55 and 1.78 g.cm?3. The bean starch granules were round to oval with a smooth surface. Results viscoamylographic profiles of the starches and flours showed the possibility of selecting cultivars for specific applications according to these characteristics.