Effects of sequential enzymatic hydrolysis on structural, bioactive and functional properties of Phaseolus lunatus protein isolate

Significant initiatives exist within the global food market to search for new, alternative protein sources with better technological, functional, and nutritional properties. Lima bean (Phaseolus lunatus L.) protein isolate was hydrolyzed using a sequential pepsin-pancreatin enzymatic system. Hydrolysis was performed to produce limited (LH) and extensive hydrolysate (EH), each with different degrees of hydrolysis (DH). The effects of hydrolysis were evaluated in vitro in both hydrolysates based on structural, functional and bioactive properties. Structural properties analyzed by electrophoretic profile indicated that LH showed residual structures very similar to protein isolate (PI), although composed of mixtures of polypeptides that increased hydrophobic surface and denaturation temperature. Functionality of LH was associated with amino acid composition and hydrophobic/hydrophilic balance, which increased solubility at values close to the isoelectric point. Foaming and emulsifying activity index values were also higher than those of PI. EH showed a structure composed of mixtures of polypeptides and peptides of low molecular weight, whose intrinsic hydrophobicity and amino acid profile values were associated with antioxidant capacity, as well as inhibiting angiotensin-converting enzyme. The results obtained indicated the potential of Phaseolus lunatus hydrolysates to be incorporated into foods to improve techno-functional properties and impart bioactive properties.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Rooting cuttings of Syringa vulgaris cv. Charles Joly and Corylus avellana cv. Aurea: the influence of stock plant pruning and shoot growth

The relationship between shoot growth and rooting was examined in two, 'difficult-to root' amenity trees, Syringa vulgaris L. cv. Charles Joly and Corylus avellana L. cv. Aurea. A range of treatments reflecting severity of pruning was imposed on field-grown stock prior to bud break. To minimise variation due to the numbers of buds that developed under different treatments, bud number was restricted to 30 per plant. Leafy cuttings were harvested at different stages of the active growth phase of each species. With Syringa, rooting decreased with later harvests, but loss of rooting potential was delayed in cuttings collected from the most severe pruning treatment. Rooting potential was associated with the extent of post-excision shoot growth on the cutting but regression analyses indicated that this relationship could not entirely explain the loss of rooting with time, nor the effects due to pruning. Similarly, in Corylus rooting was promoted by severe pruning, but the relationship between apical growth on the cutting and rooting was weaker than in Syringa, and only at the last harvest did growth play a critical role in determining rooting. Another unusual factor of the last harvest of Corylus was a bimodal distribution of roots per cutting, with very few rooted cuttings having less than five roots. This implies that, for this harvest at least, the potential of an individual cutting to root is probably not limited by the number of potential rooting sites.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

In planta conditions induce genomic changes in Pseudomonas syringae pv. phaseolicola

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Plant resistance can impose stress on invading pathogens that can lead to, and select for, beneficial changes in the bacterial genome. The Pseudomonas syringae pv. phaseolicola (Pph) genomic island PPHGI-1 carries an effector gene, avrPphB (hopAR1), which triggers the hypersensitive reaction in bean plants carrying the R3 resistance gene. Interaction between avrPphB and R3 generates an antimicrobial environment within the plant, resulting in the excision of PPHGI-1 and its loss from the genome. The loss of PPHGI-1 leads to the generation of a Pph strain able to cause disease in the plant. In this study, we observed that lower bacterial densities inoculated into resistant bean (Phaseolus vulgaris) plants resulted in quicker PPHGI-1 loss from the population, and that loss of the island was strongly influenced by the type of plant resistance encountered by the bacteria. In addition, we found that a number of changes occurred in the bacterial genome during growth in the plant, whether or not PPHGI-1 was lost. We also present evidence that the circular PPHGI-1 episome is able to replicate autonomously when excised from the genome. These results shed more light onto the plasticity of the bacterial genome as it is influenced by in planta conditions.

Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel

Pseudomonas syringae pv. phaseolicola causes halo blight of the common bean, Phaseolus vulgaris, worldwide and remains difficult to control. Races of the pathogen cause either disease symptoms or a resistant hypersensitive response on a series of differentially reacting bean cultivars. The molecular genetics of the interaction between P. syringae pv. phaseolicola and bean, and the evolution of bacterial virulence, have been investigated in depth and this research has led to important discoveries in the field of plant-microbe interactions. In this review, we discuss several of the areas of study that chart the rise of P. syringae pv. phaseolicola from a common pathogen of bean plants to a molecular plant-pathogen supermodel bacterium. Taxonomy: Bacteria; Proteobacteria, gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas; species Pseudomonas syringae; Genomospecies 2; pathogenic variety phaseolicola. Microbiological properties: Gram-negative, aerobic, motile, rod-shaped, 1.5 µm long, 0.7-1.2 µm in diameter, at least one polar flagellum, optimal temperatures for growth of 25-30 °C, oxidase negative, arginine dihydrolase negative, levan positive and elicits the hypersensitive response on tobacco. Host range: Major bacterial disease of common bean (Phaseolus vulgaris) in temperate regions and above medium altitudes in the tropics. Natural infections have been recorded on several other legume species, including all members of the tribe Phaseoleae with the exception of Desmodium spp. and Pisum sativum. Disease symptoms: Water-soaked lesions on leaves, pods, stems or petioles, that quickly develop greenish-yellow haloes on leaves at temperatures of less than 23 °C. Infected seeds may be symptomless, or have wrinkled or buttery-yellow patches on the seed coat. Seedling infection is recognized by general chlorosis, stunting and distortion of growth. Epidemiology: Seed borne and disseminated from exudation by water-splash and wind occurring during rainfall. Bacteria invade through wounds and natural openings (notably stomata). Weedy and cultivated alternative hosts may also harbour the bacterium. Disease control: Some measure of control is achieved with copper formulations and streptomycin. Pathogen-free seed and resistant cultivars are recommended. Useful websites: Pseudomonas-plant interaction http://www.pseudomonas-syringae.org/; PseudoDB http://xbase.bham.ac.uk/pseudodb/; Plant Associated and Environmental Microbes Database (PAMDB) http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; PseudoMLSA Database http://www.uib.es/microbiologiaBD/Welcome.html.

Investigation of the hypothalamic defensive system in the mouse

The hypothalamus plays especially important roles in various endocrine, autonomic, and behavioral responses that guarantee the survival of both the individual and the species. In the rat, a distinct hypothalamic defensive circuit has been defined as critical for integrating predatory threats, raising an important question as to whether this concept could be applied to other prey species. To start addressing this matter, in the present study, we investigated, in another prey species (the mouse), the pattern of hypothalamic Fos immunoreactivity in response to exposure to a predator (a rat, using the Rat Exposure Test). During rat exposure, mice remained concealed in the home chamber for a longer period of time and increased freezing and risk assessment activity. We were able to show that the mouse and the rat present a similar pattern of hypothalamic activation in response to a predator. Of particular note, similar to what has been described for the rat, we observed in the mouse that predator exposure induces a striking activation in the elements of the medial hypothalamic defensive system, namely, the anterior hypothalamic nucleus, the dorsomedial part of the ventromedial hypothalamic nucleus and the dorsal premammillary nucleus. Moreover, as described for the rat, predator-exposed mice also presented increased Fos levels in the autonomic and parvicellular parts of the paraventricular hypothalamic nucleus, lateral preoptic area and subfornical region of the lateral hypothalamic area. In conclusion, the present data give further support to the concept that a specific hypothalamic defensive circuit should be preserved across different prey species. (C) 2008 Elsevier B.V. All rights reserved.

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats (Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 +/- 1 degrees C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia. respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem. (C) 2011 Elsevier B.V. All rights reserved.

Organization of ventral tegmental area projections to the ventral tegmental area-nigral complex in the rat

The ventral tegmental area (VTA) is a nodal link in reward circuitry. Based on its striatal output, it has been subdivided in a caudomedial part which targets the ventromedial striatum, and a lateral part which targets the ventrolateral striatum [Ikemoto S (2007) Dopamine reward circuitry: two projection systems from the ventral midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res Rev 56:27-78]. Whether these two VTA parts are interconnected and to what extent the VTA innervates the substantia nigra compacta (SNc) and retrorubral nucleus (RR) are critical issues for understanding information processing in the basal ganglia. Here, VTA projections to the VTA-nigral complex were examined in rats, using Phaseolus vulgaris leucoagglutinin (PHA-L) as anterograde tracer. The results show that the dorsolateral VTA projects to itself, as well as to the dorsal tier of the SNc and RR, largely avoiding the caudomedial VTA. The ventrolateral VTA innervates mainly the interfascicular nucleus. The components of the caudomedial VTA (the interfascicular, paranigral and caudal linear nuclei) are connected with each other. In addition, the caudomedial VTA (especially the paranigral and caudal linear nuclei) innervates the lateral VTA, and, to a lesser degree, the SNc and RR. The caudal pole of the VTA sends robust, bilateral projections to virtually all the VTA-nigral complex, which terminate in the dorsal and ventral tiers. Modest inputs from the medial supramammillary nucleus to ventromedial parts of the VTA-nigral complex were also identified. In double-immunostained sections, PHA-L-labeled varicosities were sometimes found apposed to tyrosine hydroxylase-positive neurons in the ventral mesencephalon. Overall, the results underscore that VTA projections to the VTA-nigral complex are substantial and topically organized. In general, these projections, like the spiralated striato-nigro-striatal loops, display a medial-to-lateral organization. This anatomical arrangement conceivably permits the ventromedial striatum to influence the activity of the lateral striatum. The caudal pole of the VTA appears to be a critical site for a global recruitment of the mesotelencephalic system. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Interferência das plantas daninhas no feijoeiro carioca

Este trabalho foi realizado com o objetivo de determinar os períodos de interferência das plantas daninhas na cultura do feijoeiro (Phaseolus vulgaris) plantado em janeiro. A semeadura do cv. Carioca foi feita no sistema convencional e os tratamentos constaram de dois grupos: no primeiro, a cultura do feijão permaneceu livre da interferência das plantas daninhas desde a emergência até 10, 20, 30, 40, 50, 60 e 70 dias (todo o ciclo da cultura); no segundo, a cultura permaneceu sob interferência desde a semeadura até os mesmos períodos descritos anteriormente, totalizando assim 14 tratamentos. O delineamento experimental utilizado foi o de blocos casualizados com quatro repetições. A comunidade infestante foi composta por 13 espécies, com Alternanthera tenella, Blaenvillea rhomboidea e Cenchrus echinatus se destacandodas demais, representando 63,4% do total de indivíduos. O período anterior à interferência (PAI) ocorreu até os 17 dias após emergência da cultura, e o período total de prevenção à interferência (PTPI) ocorreu até 25 dias após a emergência da cultura. A interferência das plantas daninhas durante todo o ciclo de vida do feijoeiro reduziu-lhe a produtividade em 67%.

Comunidade infestante e sua interferência no feijoeiro implantado sob plantio direto, na safra de primavera

O objetivo deste estudo foi determinar os períodos críticos de interferência das plantas daninhas no feijoeiro no sistema de plantio direto, na safra de primavera. A cultura antecessora ao feijoeiro foi a de milheto, que gerou 2 t ha-1 de massa seca. Os tratamentos foram constituídos por períodos crescentes (0, 10, 17, 24, 31, 45 e 66 dias após a emergência) de convivência ou de controle das plantas daninhas. O delineamento experimental adotado foi o de blocos ao acaso, com os tratamentos dispostos no esquema fatorial 2 x 6, com quatro repetições, mais duas testemunhas: uma limpa durante todo o ciclo da cultura e outra suja, ausente de controle. A comunidade infestante foi composta por 16 espécies, sendo dez eudicotiledôneas. A espécie de maior importância foi a losna-branca (Parthenium hysterophorus). O período anterior à interferência (PAI) ocorreu até o estádio fenológico V2, e o período total de prevenção à interferência (PTPI), até o estádio V4; assim, o período crítico de prevenção à interferência (PCPI) ficou entre os estádios V2 e V4 do feijoeiro. A safra de primavera mostrou-se alternativa viável de produção. O acúmulo médio de massa e o número médio de indivíduos das plantas daninhas foram pequenos, ocasionando menor interferência na cultura e, consequentemente, um curto PCPI. Houve redução de 19% na produtividade média do feijoeiro quando a cultura conviveu durante todo o seu ciclo com a comunidade infestante.

Efeito da densidade e da distância de caruru-de-mancha e amendoim-bravo na cultura do feijoeiro

O objetivo deste trabalho foi avaliar a interferência causada pelo caruru-demancha (Amaranthus viridis) e amendoim-bravo (Euphorbia heterophylla), em função das densidades e distâncias, no feijoeiro (Phaseolus vulgaris) cultivar Pérola. Como recipientes, foram utilizadas caixas de cimento-amianto, com capacidade para 50 litros, preenchidas com LatossoloVermelho-Escuro. As mudas foram formadas em bandejas de 128 células preenchidas com substrato hortícola; quando as plântulas atingiram o estádio V2, foram transplantadas para as caixas, sendo as de feijoeiro numa linha central, reproduzindo a semeadura em campo, e as das plantas daninhas nas densidades de 8, 16 e 32 plantas m-2, distanciadas de 0, 12 e 24 cm das plantas de feijão e igualmente entre si. O experimento foi conduzido no delineamento experimental de blocos casualizados, com os tratamentos dispostos em esquema fatorial 3x3+2T, com quatro repetições, constituindo as parcelas experimentais. Foram avaliadas características de crescimento e de produtividade da cultura e das plantas daninhas. Os dados obtidos foram submetidos à análise de variância pelo teste F, e as médias, comparadas pelo teste de Tukey. Observou-se que as plantas daninhas obtiveram maior desenvolvimento quando em maior distância da cultura. O caruru-de-mancha causou reduções no número de vagens e na produtividade estimada do feijoeiro. Para o caruru-de-mancha, o aumento da densidade só causou redução na produtividade da cultura quando as plantas estavam distanciadas em pelo menos 12 cm. A 0 cm, o feijoeiro tornou-se mais competitivo e não sofreu interferência das plantas daninhas, independentemente da densidade destas.

Influencia de las malezas sobre el cultivo de frijol en función de espaciamiento y de la densidad de plantas

La hipótesis de investigación es que con la reducción de la densidad de siembra y del espaciamiento entre líneas, el período anterior a la interferencia de las malezas será reducido en el cultivo de frijol. El objetivo de este trabajo fue determinar el período anterior a la interferencia de las malezas (PAI) en función del espaciamiento entre líneas y de su densidad poblacional. Los tratamientos fueron constituidos de ocho períodos de convivencia del cultivo con las malezas: 0-10, 0-20, 0-30, 0-40, 0-50, 0-60, 0-70 y 0-80 días después de la emergencia y un control libre de interferencia. Los períodos de convivencia fueron aplicados en dos experimentos, utilizando dos distancias entre líneas, de 0,45 y 0,60 m y en dos densidades de plantas por línea de siembra, de 10 y 15 plantas por metro. El delineamiento experimental utilizado fue en bloques completos al azar, con cuatro repeticiones. Hubo reducción de 16, 40, 36 y 58% en la productividad de granos del cultivo de frijol cuando convivió durante todo el ciclo del cultivo con las malezas, para el espaciamiento de 0,45 m en las densidades de 10 y 15 plantas m-1; y espaciamiento de 0,60 m y densidades de 10 y 15 plantas m-1, respectivamente. La productividad de granos pasó a ser afectada negativamente a partir de 28, 26, 22 y 14 días después de la emergencia, constituyéndose en los períodos anteriores a la interferencia del cultivo, respectivamente.

Modeling of weeds interference periods in bean

Objetivou-se com este trabalho determinar o período de convivência anterior à interferência das plantas daninhas (PAI) e o período anterior ao dano no rendimento econômico (PADRE) na cultura do feijão, em diferentes espaçamentos (0,45 e 0,60 m) e densidade de plantas (10 e 15 plantas m-1). Os tratamentos foram constituídos de períodos de convivência entre a cultura e as plantas daninhas (0 a 10, 0 a 20, 0 a 30, 0 a 40, 0 a 50, 0 a 60, 0 a 70 e 0 a 80 dias), mais uma testemunha sem convívio com as plantas daninhas. Adotou-se o delineamento experimental de blocos casualizados, com quatro repetições. Os períodos anteriores à interferência (PAI) da cultura foram de 23, 27, 13 e 19 dias após emergência, e os períodos anteriores ao dano no rendimento econômico (PADRE), de 10, 9, 8 e 8 dias, para os tratamentos com espaçamento de 0,45 m e densidades de semeadura de 10 e 15 plantas m-1 e para aqueles com espaçamento de 0,60 m e densidades de semeadura de 10 e 15 plantas m-1, respectivamente, o que reduziu a produtividade de grãos em 63, 50, 42 e 57%, respectivamente, com a presença das plantas daninhas durante todo o ciclo do feijoeiro.

Interferencia de malezas en el cultivo de frijol en dos sistemas de labranzas

Con este trabajo, se busco determinar el período anterior a la interferencia de las malezas que conviven con el cultivo de frijol, en ausencia o presencia de residuos vegetales de Crotalaria juncea. Los tratamientos estaban constituidos de siete períodos de convivencia del cultivo con las malezas: 0-10, 0-20, 0-30, 0-40, 0-50, 0-60 y 0-70 días después de la emergencia y también de un testigo sin presencia de las malezas en ausencia o presencia de crotalaria. El diseño experimental utilizado fue de bloques completos seleccionados al azar, con cuatro repeticiones por tratamiento. Hubo reducciones de 40,6% y 55,1% en la productividad de granos de frijol en convivencia con malezas durante todo el ciclo del cultivo, en ausencia y presencia de los residuos vegetales, respectivamente. Se concluyó que la productividad de granos fue afectada negativamente a partir de 29 y 38 días después de la emergencia, sin y con residuos, respectivamente, y eso constituyó los períodos anteriores a la interferencia.