Cycling Dof Factors: Molecular and functional characterization of Arabidopsis thaliana AtCDF3 and tomato (Solanum lycopersicum L.) SlCDF3 in response to abiotic stress

El tomate (Solanum lycopersicum L.) es considerado uno de los cultivos hortícolas de mayor importancia económica en el territorio Español. Sin embargo, su producción está seriamente afectada por condiciones ambientales adversas como, salinidad, sequía y temperaturas extremas. Para resolver los problemas que se presentan en condiciones de estrés, se han empleado una serie de técnicas culturales que disminuyen sus efectos negativos, siendo de gran interés el desarrollo de variedades tolerantes. En este sentido la obtención y análisis de plantas transgénicas, ha supuesto un avance tecnológico, que ha facilitado el estudio y la evaluación de genes seleccionados en relación con la tolerancia al estrés. Estudios recientes han mostrado que el uso de genes reguladores como factores de transcripción (FTs) es una gran herramienta para obtener nuevas variedades de tomate con mayor tolerancia a estreses abióticos. Las proteínas DOF (DNA binding with One Finger) son una familia de FTs específica de plantas (Yangisawa, 2002), que están involucrados en procesos fisiológicos exclusivos de plantas como: asimilación del nitrógeno y fijación del carbono fotosintético, germinación de semilla, metabolismo secundario y respuesta al fotoperiodo pero su preciso rol en la tolerancia a estrés abiótico se desconoce en gran parte. El trabajo descrito en esta tesis tiene como objetivo estudiar genes reguladores tipo DOF para incrementar la tolerancia a estrés abiotico tanto en especies modelo como en tomate. En el primer capítulo de esta tesis se muestra la caracterización funcional del gen CDF3 de Arabidopsis, así como su papel en la respuesta a estrés abiótico y otros procesos del desarrollo. La expresión del gen AtCDF3 es altamente inducido por sequía, temperaturas extremas, salinidad y tratamientos con ácido abscísico (ABA). La línea de inserción T-DNA cdf3-1 es más sensible al estrés por sequía y bajas temperaturas, mientras que líneas transgénicas de Arabidopsis 35S::AtCDF3 aumentan la tolerancia al estrés por sequía, osmótico y bajas temperaturas en comparación con plantas wild-type (WT). Además, estas plantas presentan un incremento en la tasa fotosintética y apertura estomática. El gen AtCDF3 se localiza en el núcleo y que muestran una unión específica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activación transcripcional en ensayos de protoplastos de Arabidopsis. El dominio C-terminal de AtCDF3 es esencial para esta localización y su capacidad activación, la delección de este dominio reduce la tolerancia a sequía en plantas transgénicas 35S::AtCDF3. Análisis por microarray revelan que el AtCDF3 regula un set de genes involucrados en el metabolismo del carbono y nitrógeno. Nuestros resultados demuestran que el gen AtCDF3 juega un doble papel en la regulación de la respuesta a estrés por sequía y bajas temperaturas y en el control del tiempo de floración. En el segundo capítulo de este trabajo se lleva a cabo la identificación de 34 genes Dof en tomate que se pueden clasificar en base a homología de secuencia en cuatro grupos A-D, similares a los descritos en Arabidopsis. Dentro del grupo D se han identificado cinco genes DOF que presentan características similares a los Cycling Dof Factors (CDFs) de Arabidopsis. Estos genes son considerados ortólogos de Arabidopsis CDF1-5, y han sido nombrados como Solanum lycopersicum CDFs o SlCDFs. Los SlCDF1-5 son proteínas nucleares que muestran una unión específica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activación transcripcional in vivo. Análisis de expresión de los genes SlCDF1-5 muestran diferentes patrones de expresión durante el día y son inducidos de forma diferente en respuesta a estrés osmótico, salino, y de altas y bajas temperaturas. Plantas de Arabidopsis que sobre-expresan SlCDF1 y SlCDF3 muestran un incremento de la tolerancia a la sequía y salinidad. Además, de la expresión de varios genes de respuesta estrés como AtCOR15, AtRD29A y AtERD10, son expresados de forma diferente en estas líneas. La sobre-expresión de SlCDF3 en Arabidopsis promueve un retardo en el tiempo de floración a través de la modulación de la expresión de genes que controlan la floración como CONSTANS (CO) y FLOWERING LOCUS T (FT). En general, nuestros datos demuestran que los SlCDFs están asociados a funciones aun no descritas, relacionadas con la tolerancia a estrés abiótico y el control del tiempo de floración a través de la regulación de genes específicos y a un aumento de metabolitos particulares. ABSTRACT Tomato (Solanum lycopersicum L.) is one of the horticultural crops of major economic importance in the Spanish territory. However, its production is being affected by adverse environmental conditions such as salinity, drought and extreme temperatures. To resolve the problems triggered by stress conditions, a number of agricultural techniques that reduce the negative effects of stress are being frequently applied. However, the development of stress tolerant varieties is of a great interest. In this direction, the technological progress in obtaining and analysis of transgenic plants facilitated the study and evaluation of selected genes in relation to stress tolerance. Recent studies have shown that a use of regulatory genes such as transcription factors (TFs) is a great tool to obtain new tomato varieties with greater tolerance to abiotic stresses. The DOF (DNA binding with One Finger) proteins form a family of plant-specific TFs (Yangisawa, 2002) that are involved in the regulation of particular plant processes such as nitrogen assimilation, photosynthetic carbon fixation, seed germination, secondary metabolism and flowering time bur their precise roles in abiotic stress tolerance are largely unknown. The work described in this thesis aims at the study of the DOF type regulatory genes to increase tolerance to abiotic stress in both model species and the tomato. In the first chapter of this thesis, we present molecular characterization of the Arabidopsis CDF3 gene as well as its role in the response to abiotic stress and in other developmental processes. AtCDF3 is highly induced by drought, extreme temperatures, salt and abscisic acid (ABA) treatments. The cdf3-1 T-DNA insertion mutant was more sensitive to drought and low temperature stresses, whereas the AtCDF3 overexpression enhanced the tolerance of transgenic plants to drought, cold and osmotic stress comparing to the wild-type (WT) plants. In addition, these plants exhibit increased photosynthesis rates and stomatal aperture. AtCDF3 is localized in the nuclear region, displays specific binding to the canonical DNA target sequences and has a transcriptional activation activity in Arabidopsis protoplast assays. In addition, the C-terminal domain of AtCDF3 is essential for its localization and activation capabilities and the deletion of this domain significantly reduces the tolerance to drought in transgenic 35S::AtCDF3 overexpressing plants. Microarray analysis revealed that AtCDF3 regulated a set of genes involved in nitrogen and carbon metabolism. Our results demonstrate that AtCDF3 plays dual roles in regulating plant responses to drought and low temperature stress and in control of flowering time in vegetative tissues. In the second chapter this work, we carried out to identification of 34 tomato DOF genes that were classified by sequence similarity into four groups A-D, similar to the situation in Arabidopsis. In the D group we have identified five DOF genes that show similar characteristics to the Cycling Dof Factors (CDFs) of Arabidopsis. These genes were considered orthologous to the Arabidopsis CDF1 - 5 and were named Solanum lycopersicum CDFs or SlCDFs. SlCDF1-5 are nuclear proteins that display specific binding to canonical DNA target sequences and have transcriptional activation capacities in vivo. Expression analysis of SlCDF1-5 genes showed distinct diurnal expression patterns and were differentially induced in response to osmotic, salt and low and high temperature stresses. Arabidopsis plants overexpressing SlCDF1 and SlCDF3 showed increased drought and salt tolerance. In addition, various stress-responsive genes, such as AtCOR15, AtRD29A and AtERD10, were expressed differently in these lines. The overexpression of SlCDF3 in Arabidopsis also results in the late flowering phenotype through the modulation of the expression of flowering control genes such CONSTANS (CO) and FLOWERING LOCUS T (FT). Overall, our data connet SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites.

Report of Agricultural Experiment Station, Agricultural and Mechanical College, Auburn, Ala., October 1888.

"New Series"--Cover.

Report of Agricultural Experiment Station, Agricultural and Mechanical College, Auburn, Ala., July 1888.

Cover title.

Transactions and proceedings of the ... annual meeting of the California Association of Nurserymen ...

Mode of access: Internet.

Infection of the immature stages of Diadegma semiclausum, an endolarval parasitoid of the diamondback moth, by Beauveria bassiana

Interactions between the immature stages of Diadegma semiclausum, an endolarval parasitoid of Plutella xylostella, and the fungal entomopathogen Beauveria bassiana were investigated in the laboratory. Detrimental effects of B. bassiana on D. semiclausum cocoon production and adult parasitoid emergence increased with increasing pathogen concentration and some parasitoid larvae became infected by B. bassiana within hosts. The negative impact of B. bassiana on D. semiclausum cocoon production decreased as temporal separation between parasitism and pathogen exposure increased. Adult parasitoid emergence was significantly compromised by the highest rates of B. bassiana tested even when exposure of host larvae to the pathogen was delayed until one day before predicted parasitoid cocoon formation. Parasitoid pupae were infected by the pathogen in all B. bassiana treatments which did not preclude their development. (C) 2004 Elsevier Inc. All rights reserved.

Initiation of fungal epizootics in diamondback moth populations within a large field cage: proof of concept for auto-dissemination

Adult diamondback moths (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), inoculated with the fungus Zoophthora radicans, were released within a large field cage containing DBM-infested potted broccoli plants. Larvae and pupae on exposed and caged control plants were examined on five occasions over the next 48 days for evidence of Z. radicans infection. Infected larvae were first detected on exposed plants 4 days after the initial release of adults, and after 48 days the infection level reached 79%. Aerially borne conidia were a factor in transmission of the fungus. Infection had no effect on possible losses of larval and adult cadavers due to scavengers in field crops. In a trial to measure the influence of infection on dispersal, twice as many non-infected as infected males were recaptured in pheromone traps, although the difference in cumulative catch only became significant 3 days after release of the males. In a separate experiment, when adult moths were inoculated with Beauveria bassiana conidia and released into the field cage, DBM larvae collected from 37 of 96 plants sampled 4 days later subsequently died from B. bassiana infection. The distribution of plants from which the infected larvae were collected was random, but the distribution of infected larvae was clustered within the cage. These findings suggest that the auto-dissemination of fungal pathogens may be a feasible strategy for DBM control, provided that epizootics can be established and maintained when DBM population densities are low.

Role of Diadegma semiclausum (Hymenoptera: Ichneumonidae) in controlling Plutella xylostella (Lepidoptera: Plutellidae): Cage exclusion experiments and direct observation

We evaluated the role of the larval parasitoid, Diadegma semiclausum Hellen (Hymenoptera: Ichneumonidae), in controlling Plutella xylostella (L.) (Lepidoptera: Plutellidae) by cage exclusion experiments and direct field observation during the winter season in southern Queensland, Australia. The cage exclusion experiment involved uncaged, open cage and closed cage treatments. A higher percentage (54-83%) of P. xylostella larvae on sentinel plants were lost in the uncaged treatment than the closed (4-9%) or open cage treatments (11-29%). Of the larvae that remained in the uncaged treatment, 72-94% were parasitized by D. semiclausum , much higher than that in the open cage treatment (8-37% in first trial, and 38-63% in second trial). Direct observations showed a significant aggregation response of the field D. semiclausum populations to high host density plants in an experimental plot and to high host density plots that were artificially set-up near to the parasitoid source fields. The degree of aggregation varied in response to habitat quality of the parasitoid source field and scales of the manipulated host patches. As a result, density-dependence in the pattern of parasitism may depend on the relative degree of aggregation of the parasitoid population at a particular scale. A high degree of aggregation seems to be necessary to generate density-dependent parasitism by D. semiclausum . Integration of the cage exclusion experiment and direct observation demonstrated the active and dominant role of this parasitoid in controlling P. xylostella in the winter season. A biologically based IPM strategy, which incorporates the use of D. semiclausum with Bt, is suggested for the management of P. xylostella in seasons or regions with a mild temperature.

Modelling the population dynamics of the Queensland fruit fly, Bactrocera (Dacus) tryoni: a cohort-based approach incorporating the effects of weather

Queensland fruit fly, Bactrocera (Dacus) tryoni (QFF) is arguably the most costly horticultural insect pest in Australia. Despite this, no model is available to describe its population dynamics and aid in its management. This paper describes a cohort-based model of the population dynamics of the Queensland fruit fly. The model is primarily driven by weather variables, and so can be used at any location where appropriate meteorological data are available. In the model, the life cycle is divided into a number of discreet stages to allow physiological processes to be defined as accurately as possible. Eggs develop and hatch into larvae, which develop into pupae, which emerge as either teneral females or males. Both females and males can enter reproductive and over-wintering life stages, and there is a trapped male life stage to allow model predictions to be compared with trap catch data. All development rates are temperature-dependent. Daily mortality rates are temperature-dependent, but may also be influenced by moisture, density of larvae in fruit, fruit suitability, and age. Eggs, larvae and pupae all have constant establishment mortalities, causing a defined proportion of individuals to die upon entering that life stage. Transfer from one immature stage to the next is based on physiological age. In the adult life stages, transfer between stages may require additional and/or alternative functions. Maximum fecundity is 1400 eggs per female per day, and maximum daily oviposition rate is 80 eggs/female per day. The actual number of eggs laid by a female on any given day is restricted by temperature, density of larva in fruit, suitability of fruit for oviposition, and female activity. Activity of reproductive females and males, which affects reproduction and trapping, decreases with rainfall. Trapping of reproductive males is determined by activity, temperature and the proportion of males in the active population. Limitations of the model are discussed. Despite these, the model provides a useful agreement with trap catch data, and allows key areas for future research to be identified. These critical gaps in the current state of knowledge exist despite over 50 years of research on this key pest. By explicitly attempting to model the population dynamics of this pest we have clearly identified the research areas that must be addressed before progress can be made in developing the model into an operational tool for the management of Queensland fruit fly. (C) 2003 Published by Elsevier B.V.

Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control

Biological control is the purposeful introduction of parasites, predators, and pathogens to reduce or suppress pest populations. Wolbachia are inherited bacteria of arthropods that have recently attracted attention for their potential as new biocontrol agents. Wolbachia manipulate host reproduction by using several strategies, one of which is cytoplasmic incompatibility (CI) [Stouthamer, R., Breeuwer, J. A. J. & Hurst, G. D. D. (1999) Annu. Rev. Microbiol. 53,71-102]. We established Wolbachia-infected lines of the medfly Ceratitis capitata using the infected cherry fruit fly Rhagoletis cerasi as donor. Wolbachia induced complete CI in the novel host. Laboratory cage populations were completely suppressed by single releases of infected males, suggesting that Wolbachia-induced CI could be used as a novel environmentally friendly tool for the control of medfly populations. The results also encourage the introduction of Wolbachia into pest and vector species of economic and hygenic relevance to suppress or modify natural populations.

Application of microencapsulated flavor to extrusion product

Flavoring is still a difficult problem in the snack food industry because of the high volatility of flavors and their instability under extrusion condition. Although postextrusion added flavor is commonly used, it suffers from numerous drawbacks. Flavor losses at the exit die because flash distillation is a critical issue and can only be minimized by controlling the pressure difference at the end of the barrel and the exit die, which, however, affects other desirable product characteristics. Residence time distribution (RTD), as an important intermediate process variable that among others controls the extent of reactions, can also be a major determinant on flavor retention during extrusion. Encapsulation of flavors is a promising alternative to enhance the retention of preextrusion added flavor during extrusion. The capsules should withstand high temperature and shear conditions in, the extruder barrel. Various encapsulation techniques and their encapsulated flavor characteristics are illustrated.

The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria

The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and poly-L-lysine in combination with alginate. The thickness of the alginate beads increased with the addition of coating materials. No differences were detectable in the bead strength by texture analysis or in the thickness of the beads with different types of coating materials by transmission electron microscopy. The survivability of three probiotics in uncoated beads, coated beads, and as free cells (unencapsulated) was conducted in 0.6% bile salt solution and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated heads. (C) 2004 Elsevier Ltd. All rights reserved.

Stickiness measurement techniques for food powders: a review

The importance of sticky behaviour of amorphous food powders has been recognized over many decades in the food industry due to its influence on process and handling abilities and quality of the powders. This paper emphasizes the role of stickiness in the food powder industry as well as reviews the stickiness characterization techniques developed to date. This paper also attempts to correlate the stickiness behaviour of food powders to the instrumental analysis such as glass transition temperature. (C) 2004 Elsevier B.V All rights reserved.

Effect of high power ultrasound waves on properties of meat: A review

The effect of high power ultrasound waves on physical, biochemical, and microbial properties of meat have been the subject of a great deal of interest in recent years. The present review details the basic principles underlying the effects of ultrasound on the properties of food systems, followed by discussion of specific effects of high power ultrasound on meat products, including muscle, cellular, and subcellular components. In addition, the specific effects of high power ultrasound on the following parameters are discussed: enzyme activities and efficiencies, muscle proteolysis, quality criteria such as tenderness; extraction of protein, gelation, and restructuring of meat products and germicidal properties against meat micro-organisms.

Effect of addition of maltodextrin on drying kinetics and stickiness of sugar and acid-rich foods during convective drying: experiments and modelling

The effect of addition of maltodextrin on drying kinetics of drops containing fructose, glucose, sucrose and citric acid individually and in mixtures was studied experimentally using single drop drying experiments and numerically by solving appropriate mass and heat transfer equations. The numerical predictions agreed with the experimental moisture and temperature histories within 5-6% average relative (absolute) errors and average differences of +/- 1degreesC, respectively. The stickiness of these drops was determined using the glass transition temperature (T-g) of the drops' surface layer as an indicator. The experimental stickiness histories followed the model predictions with reasonable accuracy. A safe drying (non-sticky) regime in a spray drying environment has been proposed, and used to estimate the optimum amount of addition of maltodextrin for successful spray drying of 120 micron diameter droplets of fruit juices. (C) 2003 Elsevier Ltd. All rights reserved.