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.

Evaluation of the influence of visual parameters on wave transmission velocity in sawn chestnut timber

Non-destructive, visual evaluation and mechanical testing techniques were used to assess the structural properties of 374 samples of chestnut (Castanea sativa). The principal components method was applied to establish and interpret correlations between variables obtained of modulus of elasticity, bending strength and density. The static modulus of elasticity presented higher correlation values than those obtained using non-destructive methods. Bending strength presented low correlations with the non-destructive parameters, but there was some relation to the different knot ratios defined. The relationship was stronger with the most widely used ratio, CKDR. No significant correlations were observed between any of the variables and density.

Genetic characterization of chestnut (Castanea sativa Mill.) orchards and traditional nut varieties in El Bierzo, a glacial refuge and major cultivation site in Northwestern Spain

This paper presents a detailed genetic study of Castanea sativa in El Bierzo, a major nut production region with interesting features. It is located within a glacial refuge at one extreme of the distribution area (northwest Spain); it has a centenary tradition of chestnut management; and more importantly, it shows an unusual degree of genetic isolation. Seven nuclear microsatellite markers were selected to analyze the genetic variability and structure of 169 local trees grafted for nut production. We analyzed in the same manner 62 local nuts. The selected loci were highly discriminant for the genotypes studied, giving a combined probability of identity of 6.1 × 10−6. An unprecedented density of trees was sampled for this project over the entire region, and nuts were collected representing 18 cultivars marketed by local producers. Several instances of misclassification by local growers were detected. Fixation index estimates and analysis of molecular variance (AMOVA) data are supportive of an unexpectedly high level of genetic differentiation in El Bierzo, larger than that estimated in a previous study with broader geographical scope but based on limited local sampling (Pereira-Lorenzo et al., Tree Genet Genomes 6: 701–715, 2010a). Likewise, we have determined that clonality due to grafting had been previously overestimated. In line with these observations, no significant spatial structure was found using both a model-based Bayesian procedure and Mantel’s tests. Taken together, our results evidence the need for more fine-scale genetic studies if conservation strategies are to be efficiently improved.

Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Pasarela peatonal sobre el río Ulla, provincia de Lugo (Galicia)

Se trata de un proyecto de pasarela peatonal colgante sobre el río Ulla, de aproximadamente 36 metros de luz. La especial belleza del área natural en que se situará llevó a la opción de un diseño claro y sencillo para lograr su integración en el lugar. Se busca que la pasarela pase lo más desapercibida posible y deje el protagonismo para el entorno, el río y la naturaleza misma. La difícil accesibilidad de la zona condicionó el diseño y los procedimientos constructivos a adoptar. Se optó por una tipología de pasarela colgante de cables de acero, tablero de madera de castaño y soportes de madera laminada cilíndrica, sobre unos estribos de hormigón armado parcialmente ocultos gracias a su integración en la topografía del lugar. El diseño modular del tablero, construido a base de piezas prefabricadas de madera de castaño que se ensamblan entre sí, permite el montaje en seco mediante avance del tablero sin necesidad de cimbra. Su diseño con dos capas cruzadas de entablado solidarizadas proporciona al conjunto un comportamiento estructural similar al de una placa maciza de madera. This is the project of a suspension footbridge over the Ulla River. It has a span of about 36 meters. The beauty of the natural surroundings where it is located guided the design to the option of a clear and simple proposal with the aim of achieving an integrated project for the area. The footbridge tries to be as invisible as possible with the purpose of maintaining the natural quality of the river and its surroundings. The difficult access to the area conditioned the design and the construction procedures chosen. A suspension footbridge with stainless steel cables, chestnut timber deck and cylindrical glulam piles was designed in the end. The reinforced concrete abutments are partially concealed thanks to their integration in the topography of the area. The deck is built of prefabricated chestnut timber modules that can be joined together at the construction site and can be assembled without the necessity of scaffolding over the river. The design of the deck, with two crossed layers of chestnut boards screwed down together, permits a structural behaviour similar to a solid wooden slab.

Caracterización de la madera de Castanea sativa Mill. de procedencia española

La madera de castaño de procedencia española no está presente como material estructural en la norma europea EN 1912, y por lo tanto no está asignada a ninguna clase resistente como sí lo están otras especies españolas. Por ello, en el presente trabajo se realizó una caracterización de madera aserrada de castaño de Asturias con fines estructurales, realizando los ensayos según la norma UNE EN 408:2004, y calculando sus valores característicos según la norma UNE EN 384:2010. Los valores obtenidos permitieron asignar una clase resistente de las establecidas en la norma UNE EN 338:2010. Fueron evaluadas un total de 260 probetas de dos secciones (40x100 y 40x150 mm) siendo asignada una clase resistente D24, resultando la densidad y la resistencia a flexión los parámetros limitantes y observándose un valor de módulo de elasticidad superior al correspondiente a dicha clase. La relación entre los módulo de elasticidad longitudinal y transversal obtenidos experimentalmente fue de 10 aproximadamente, mientras que la normativa establece, como valor genérico, 16. Se observó que la singularidad de la madera que provocó un mayor porcentaje de rotura fue la presencia de nudos, presentando estas probetas un valor de resistencia significativamente menor. Chestnut timber from Spain is not included as a structural timber in the European standard EN 1912 nor is it assigned to any strength class like other Spanish species. Therefore, a characterization of structural chestnut timber from Asturias was made according to the UNE EN 408:2004, and the characteristic values were calculated according to the UNE EN 384:2010. The values obtained allowed the assignment of a strength class according to UNE EN 338:2010. 260 samples of two sections (40x100 and 40x150 mm) were tested and a D24 strength class was assinged. Density and bending strength were the limiting parameters, and the value of modulus of elasticity was higher than the values asssigned to D24. The relationship between modulus of elasticity and the shear modulus obtained was approximately equal to 10, while this value in the UNE EN 384:2010 is set at 16. The characteristic of the wood which caused a higher percentage of failure was the presence of knots, resulting in significantly lower resistance values.

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited mayor que50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.