Starch distribution in pear tree organs in relation to training systems, rootstocks and fruit quality

Starch is the main form in which plants store carbohydrates reserves, both in terms of amounts and distribution among different plant species. Carbohydrates are direct products of photosynthetic activity, and it is well know that yield efficiency and production are directly correlated to the amount of carbohydrates synthesized and how these are distributed among vegetative and reproductive organs. Nowadays, in pear trees, due to the modernization of orchards, through the introduction of new rootstocks and the development of new training systems, the understanding and the development of new approaches regarding the distribution and storage of carbohydrates, are required. The objective of this research work was to study the behavior of carbohydrate reserves, mainly starch, in different pear tree organs and tissues: i.e., fruits, leaves, woody organs, roots and flower buds, at different physiological stages during the season. Starch in fruit is accumulated at early stages, and reached a maximum concentration during the middle phase of fruit development; after that, its degradation begins with a rise in soluble carbohydrates. Moreover, relationships between fruit starch degradation and different fruit traits, soluble sugars and organic acids were established. In woody organs and roots, an interconversion between starch and soluble carbohydrates was observed during the dormancy period that confirms its main function in supporting the growth and development of new tissues during the following spring. Factors as training systems, rootstocks, types of bearing wood, and their position on the canopy, influenced the concentrations of starch and soluble carbohydrates at different sampling dates. Also, environmental conditions and cultural practices must be considered to better explain these results. Thus, a deeper understanding of the dynamics of carbohydrates reserves within the plant could provide relevant information to improve several management practices to increase crop yield efficiency.

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Minimal residual disease in cancer therapy - Small things make all the difference

Minimal residual disease (MRD) is a major hurdle in the eradication of malignant tumors. Despite the high sensitivity of various cancers to treatment, some residual cancer cells persist and lead to tumor recurrence and treatment failure. Obvious reasons for residual disease include mechanisms of secondary therapy resistance, such as the presence of mutant cells that are insensitive to the drugs, or the presence of cells that become drug resistant due to activation of survival pathways. In addition to such unambiguous resistance modalities, several patients with relapsing tumors do not show refractory disease and respond again when the initial therapy is repeated. These cases cannot be explained by the selection of mutant tumor cells, and the precise mechanisms underlying this clinical drug resistance are ill-defined. In the current review, we put special emphasis on cell-intrinsic and -extrinsic mechanisms that may explain mechanisms of MRD that are independent of secondary therapy resistance. In particular, we show that studying genetically engineered mouse models (GEMMs), which highly resemble the disease in humans, provides a complementary approach to understand MRD. In these animal models, specific mechanisms of secondary resistance can be excluded by targeted genetic modifications. This allows a clear distinction between the selection of cells with stable secondary resistance and mechanisms that result in the survival of residual cells but do not provoke secondary drug resistance. Mechanisms that may explain the latter feature include special biochemical defense properties of cancer stem cells, metabolic peculiarities such as the dependence on autophagy, drug-tolerant persisting cells, intratumoral heterogeneity, secreted factors from the microenvironment, tumor vascularization patterns and immunosurveillance-related factors. We propose in the current review that a common feature of these various mechanisms is cancer cell dormancy. Therefore, dormant cancer cells appear to be an important target in the attempt to eradicate residual cancer cells, and eventually cure patients who repeatedly respond to anticancer therapy but lack complete tumor eradication.

Césped deportivo : comportamiento del cultivar AZ-1 de Pennisetum clandestinum Hochst. ex Chiov. (kikuyo). Paraná, Entre Ríos, Argentina

A fines de 2001 se incorporó al mercado argentino el cultivar AZ-1 de Pennisetum clandestinum Hochst. ex Chiov. (kikuyo) para ser utilizado en campos deportivos o con fines ornamentales. Con el propósito de caracterizar esta variedad comercial en el departamento Paraná (provincia de Entre Ríos, Argentina) se evaluó su comportamiento y aptitud para césped ornamental y/o deportivo en condiciones de mantenimiento para césped de calidad, bajo dos condiciones de drenaje. Se evaluaron cobertura, color, textura, distancia de entrenudos, grosor de estolones y período de dormición. El ensayo se realizó en Oro Verde, departamento Paraná, en un suelo Molisol y consistió en 2 tratamientos: con y sin drenaje, con 4 repeticiones cada uno. El tamaño de la parcela fue de 2,5 por 5,0 m. El diseño experimental utilizado fue parcelas apareadas y las mediciones se realizaron desde junio hasta noviembre de 2005. Las características climáticas locales son: temperatura media anual de 18,1°C y un régimen isohigro de 947,6 mm de precipitación anual. El cultivar se comportó como apto para césped en las características y condiciones de manejo evaluadas, sin diferencias entre los tratamientos con y sin drenaje. Presentó rápida implantación, niveles altos de cobertura, color claro y uniforme, textura media y estolones gruesos a medianos a través del tiempo. En invierno no perdió cobertura ni color. Se caracterizó como un césped no apto para campos deportivos de alta exigencia, pero recomendable para clubes de bajo presupuesto y mantenimiento.

Temperaturas óptimas para resembrar especies mesotérmicas sobre céspedes de Cynodon dactylon en el centro-sur de la provincia de Córdoba, Argentina

Los objetivos fueron determinar temperaturas medias de suelo (TMS) y mínimas de aire (TMA) adecuadas para realizar resiembras otoñales; evaluar la tasa de crecimiento (TCC) de Cynodon dactylon (Bermuda) que minimice la competencia con la especie resembrada y el comportamiento de distintas especies mesotérmicas en el centro-sur de la provincia de Córdoba. Se resembró durante 2010 y 2011, en un diseño de parcelas divididas con cinco fechas de siembra (FS) quincenalmente desde principios de marzo, con tres genotipos: Lolium multiflorum cv Axcella, L. perenne y L. perenne cv Excel I. Las temperaturas se midieron entre principios de marzo y hasta que la bermuda entró en dormición, calculando TMA y TMS. En las subparcelas resembradas se registraron calidad visual y cobertura total; además, en la bermuda se calculó la TCC. Las resiembras que lograron valores más altos de calidad visual, cobertura y bajas TCC de la Bermuda, se dieron con TMS entre 18 - 21°C y TMA entre 11,5 - 15,5°C. De acuerdo con los valores medios normales de la región centro-sur de Córdoba, las temperaturas óptimas se observan entre la segunda década de marzo y primera de abril. Los tres genotipos tuvieron comportamientos similares independientemente de la FS utilizada.

Macroinvertebrate abundance and proportion of trait categories in river biotopes across England and Wales

There is a long tradition of river monitoring using macroinvertebrate communities to assess environmental quality in Europe. A promising alternative is the use of species life-history traits. Both methods, however, have relied on the time-consuming identification of taxa. River biotopes, 1-100 m**2 'habitats' with associated species assemblages, have long been seen as a useful and meaningful way of linking the ecology of macroinvertebrates and river hydro-morphology and can be used to assess hydro-morphological degradation in rivers. Taxonomic differences, however, between different rivers had prevented a general test of this concept until now. The species trait approach may overcome this obstacle across broad geographical areas, using biotopes as the hydro-morphological units which have characteristic species trait assemblages. We collected macroinvertebrate data from 512 discrete patches, comprising 13 river biotopes, from seven rivers in England and Wales. The aim was to test whether river biotopes were better predictors of macroinvertebrate trait profiles than taxonomic composition (genera, families, orders) in rivers, independently of the phylogenetic effects and catchment scale characteristics (i.e. hydrology, geography and land cover). We also tested whether species richness and diversity were better related to biotopes than to rivers. River biotopes explained 40% of the variance in macroinvertebrate trait profiles across the rivers, largely independently of catchment characteristics. There was a strong phylogenetic signature, however. River biotopes were about 50% better at predicting macroinvertebrate trait profiles than taxonomic composition across rivers, no matter which taxonomic resolution was used. River biotopes were better than river identity at explaining the variability in taxonomic richness and diversity (40% and <=10%, respectively). Detailed trait-biotope associations agreed with independent a priori predictions relating trait categories to near river bed flows. Hence, species traits provided a much needed mechanistic understanding and predictive ability across a broad geographical area. We show that integration of the multiple biological trait approach with river biotopes at the interface between ecology and hydro-morphology provides a wealth of new information and potential applications for river science and management.

Ion concentrations and extracelluar pH of Calanus glacialis in Billefjorden, Svalbard

Arctic shelf zooplankton communities are dominated by the copepod Calanus glacialis. This species feeds in surface waters during spring and summer and accumulates large amounts of lipids. Autumn and winter are spent in dormancy in deeper waters. Lipids are believed to play a major role in regulating buoyancy, however, they cannot explain fine-tuning of the depth distribution. To investigate whether ion exchange processes and acid-base regulation support ontogenetic migration as suggested for Antarctic copepods, we sampled C. glacialis in monthly intervals for 1 yr in a high-Arctic fjord and determined cation concentrations and the extracellular pH (pHe) in its hemolymph. During the winter/spring transition, prior to the upward migration of the copepods, Li+ ions were exchanged with cations (Na+, Mg2+, and Ca2+) leading to Li+ concentrations of 197 mmol/L. This likely decreased the density and promoted upward migration in C. glacialis. Our data thus suggest that Li+ has a biological function in this species. Ion and pHe regulation in the hemolymph were not directly correlated, but the pHe revealed a seasonal pattern and was low (5.5) in winter and high (7.9) in summer. Low pHe during overwintering might be related to metabolic depression and thus, support diapause.

Respiration rates and abundances of dominant copepods of the northern Benguela Current System

Respiration rates of 16 calanoid copepod species from the northern Benguela upwelling system were measured on board RRS Discovery in September/October 2010 to determine their energy requirements and assess their significance in the carbon cycle. Individual respiration rates were standardised to a mean copepod body mass and a temperature regime typical of the northern Benguela Current. These adjusted respiration rates revealed two different activity levels (active and resting) in copepodids C5 of Calanoides carinatus and females of Rhincalanus nasutus, which reduced their metabolism during dormancy by 82% and 62%, respectively. An allometric function (Imax) and an energy budget approach were performed to calculate ingestion rates. Imax generally overestimated the ingestion rates derived from the energy budget approach by >75%. We suggest that the energy budget approach is the more reliable approximation with a total calanoid copepod (mainly females) consumption of 78 mg C m-2 d-1 in neritic regions and 21 mg C m-2 d-1 in oceanic regions. The two primarily herbivorous copepods C. carinatus (neritic) and Nannocalanus minor (oceanic) contributed 83% and 5%, respectively, to total consumption by calanoid copepods. Locally, C. carinatus can remove up to 90% of the diatom biomass daily. In contrast, the maximum daily removal of dinoflagellate biomass by N. minor was 9%. These estimates imply that C. carinatus is an important primary consumers in the neritic province of the northern Benguela system, while N. minor has little grazing impact on phytoplankton populations further offshore. Data on energy requirements and total consumption rates of dominant calanoid copepods of this study are essential for the development of realistic carbon budgets and food-web models for the northern Benguela upwelling system.

Arabidopsis thaliana DOF6 negatively affects germination in non-after ripened seeds and interacts with TCP14

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system andin planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition.