Aproximaciones -ómicas al estudio de la germinación de semillas de especies recalcitrantes: el caso de la encina (Quercus ilex subsp. ballota)

La presente Tesis Doctoral está centrada en el estudio de la encina (Quercus ilex L. subsp. ballota [Desf.] Samp.), una especie dominante en los sistemas silvopastorales mediterráneos. El mantenimiento y la sostenibilidad de los bosques de encina se enfrenta actualmente con problemas y retos mayormente relacionados con la conservación de semillas viables, ya que se trata de una semilla recalcitrante, y con la alta tasa de mortalidad de los individuos adultos y plantones luego de ser trasplantados a campo, resultado de las adversas condiciones ambientales. En el presente trabajo se llevó a cabo el estudio de la germinación de semillas de encina, así como de plántulas en estadio temprano mediante una aproximación multidisciplinar que combina las técnicas clásicas de biología molecular con las “ómicas”. Con este objetivo general fueron propuestos los siguientes objetivos específicos 1. Realizar un análisis transcripcional de un grupo de genes candidatos implicados en tolerancia a la de desecación, regulación de la señalización de ABA, metabolismo y defensa contra el estrés oxidativo, a lo largo del proceso de germinación. 2. Identificar genes de expresión diferencial en semilla germinada y de plántulas muy jóvenes de Q. ilex mediante el uso de hibridación sustractiva por supresión (SSH). 3. Identificar proteínas cuya abundancia se ve alterada durante la germinación y desarrollo de plántulas en estadios tempranos, determinando los perfiles proteómicos mediante aproximaciones basadas y libres de gel. 4. Identificar el phosphoproteoma y los cambios que ocurren durante la germinación de semillas Los resultados del análisis transcripcional del grupo de genes candidatos revelaron que las semillas maduras de Q. ilex presentan algunas características fisiológicas intracelulares propias de las semillas ortodoxas que incluyen (i) acumulación de azúcares (sacarosa) y proteínas protectoras (DHN3) y (ii) acumulación de transcriptos implicados en la síntesis de oligosacáridos osmorreguladores de rafinosa (GolS) y en la defensa contra el estrés oxidativo (Sod1, Gst). Sin embargo, las semillas maduras de Q. ilex, como otras semillas recalcitrantes se dispersan con un alto contenido de agua y mantienen el metabolismo parcialmente activo. Los resultados de este análisis transcripcional fueron verificados y complementados con la determinación de (i) niveles de fitohormonas (ABA, GA3, GA4, IAA, iP and iPR), (ii) acumulación de azúcares (Sac, Glc, Fru) y (iii) determinación de proteínas mediante inmunodetección (DHN3, GAPDH, RBCL) y actividad enzimática (SOD); estos resultados mostraron una buena correlación con los transcriptos analizados. El análisis de expresión diferencial de genes mediante SSH permitió identificar por primera vez en Q. ilex un grupo de ESTs correspondiente a genes diferencialmente expresados en semillas germinadas y plántulas en desarrollo temprano. En las semillas germinadas se identificaron 31 genes sobre-expresados, entre los que se incluyen genes que codifican para proteínas implicadas en la respuesta a estreses, transporte, defensa contra estrés oxidativo, modificación de la pared celular, ciclo celular, metabolismo de proteínas y transducción. Por otro lado, 39 transcriptos no redundantes se encontraron sobre-expresados en estadio temprano de plántulas. Las proteínas codificadas por estos transcritos fueron agrupadas en siete categorías funcionales que incluyen fotosíntesis, metabolismo secundario, transporte, señalización, respuesta a diversos estreses, expresión génica y organización de componentes...This work is focused on holm oak (Quercus ilex L. subsp. ballota [Desf.] Samp.) a dominant tree species in natural forest ecosystems over large areas of the Western Mediterranean Basin. Q. ilex forest maintenance and sustainability are facing important problems and challenges related to seed viability/conservation, and plant mortality in both adult trees and young oneyear- old plants after field transplantation resulting from adverse environmental conditions such as drought and the so-called decline syndrome. In the present Doctoral Thesis, the germination and seedling growth of Q. ilex was studied applying a multidisciplinary “omics” approach combined with classical biochemical approaches, according to the actual trends in biosciences research. Within this general aim, specific objectives pursued in this thesis were: 1. Determine the expression profile of a selected group of protein coding genes involved in desiccation tolerance, regulation of ABA-signalling, metabolism and antioxidative defence. 2. Identify differentially expressed genes between germinated seeds and seedlings of Q. ilex by using a suppression subtractive hybridization (SSH). 3. Analyse changes in the proteomic profiles during germination and seedling growth through gel based and gel free approaches. 4. Analyse the dynamic protein phosphorylation changes during seed germination and seedling development. The transcriptional analysis revealed that mature Q. ilex seeds show some of the intracellular physiological characteristics of orthodox seed that included (i) accumulation of non-reducing carbohydrates (sucrose) and protective proteins (DHN3) and (ii) accumulation of transcripts involved in the synthesis of certain osmoregulator of raffinose series oligosaccharides (GolS), the anti-oxidative defence (Sod1, Gst). But the holm oak mature acorns, like other recalcitrant seeds, have the ability to maintain a partially active metabolism. The transcriptional analysis results were verified and complemented with the determination of (i) plant hormones levels (ABA; the gibberellins GA3 y GA4; the auxin IAA and the cytokinins iP and iPR,) (ii) sugars accumulation (Suc, Glc, Fru) and (iii) proteins amounts determined by immunoblotting (DHN3, GAPDH, RBCL) and/or enzymatic activity (SOD) and good correlation was found whit transcript levels. The analysis of differentially expressed genes by SSH allowed the identification for the first time a large number of putative differentially expressed ESTs from the embryo axis in germinated seeds and from shoot seedlings of Q. ilex during the postgermination and seedling establishment. Thirty-one over-expressed genes were identified in germinated seeds at the germination stage. Proteins encoded by these genes are representative of eight functional categories: stress responses, transport, oxidation-reduction, cell wall modification, cell division cycle, protein metabolism, cellular component organization and translation. On the other hand, 39 non-redundant transcripts over-represented in Q. ilex shoots seedlings were grouped in seven functional categories: photosynthesis, secondary metabolism, transport, signalling, stress response, gene expression and cellular component organization. These data constitute an important genomics resource that should clearly benefit further germination and other biological process research on Q. ilex..

Descripción del proteoma de bellota de la encina (Quercus ilex) mediante extracción secuencial de proteínas y análisis por LC-MS

Comunicaciones a congreso

Germination and Early Seedling Development in Quercus ilex Recalcitrant and Non-dormant Seeds: Targeted Transcriptional, Hormonal, and Sugar Analysis

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0–S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1–S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds

Captación de participantes en el programa experimental de prescripción de estupefacientes en Andalucía (PEPSA)

ResumenEsta nota tiene como finalidad describir los pasos seguidos en el proceso de captación de los participantes en el ensayo andaluz de prescripción de heroína intravenosa. El programa experimental de prescripción de estupefacientes en Andalucía (PEPSA) compara el tratamiento de heroína respecto a la metadona oral, en la mejoría de la salud física y mental y la integración social. Dada las características de la población diana (usuarios de heroína por vía intravenosa en situación de exclusión social para quienes no han sido efectivos los tratamientos disponibles), se planificó un abordaje específico para acercar dichas personas al estudio. Tras una investigación previa sobre la distribución de la población diana en la ciudad de Granada, se dividió ésta en 3 zonas. Se acudía a los principales centros de reunión (plazas, comedores sociales, dispensarios de metadona) y se concretaba una cita con un médico del PEPSA. El trabajo apoyado en iguales ha sido una herramienta fundamental en este proceso de captación, y ha facilitado el acercamiento a la población diana. Asimismo, este trabajo ha permitido entablar contacto con usuarios de drogas que no acceden a los servicios sociosanitarios, por lo que la labor del equipo de captación también ha sido educar en la reducción de daños y ofrecer alternativas sociales y sanitarias más allá del ensayo.AbstractIn this field note we describe the steps followed in the process of recruiting participants for the experimental drug prescription program in Andalusia (PEPSA). This trial is a comparative, randomized, open study of the difference between intravenous heroin treatment and oral methadone for socially excluded, opiate-dependent patients, in whom other available treatments have been unsuccessful. Because this is a hidden and hard-to-reach population, a specific approach was planned to put as many patients as possible in touch with the program. A previous study of the target population’s distribution in the City of Granada was performed and the city was divided into three areas. Potential participants were interviewed in squares, soup kitchens and methadone dispensaries by outreach workers and peers, who suggested they make an appointment with a PEPSA physician. Peer-driven intervention was a crucial instrument in this recruitment procedure, allowing greater access to the target population. Furthermore, this approach allowed contact with drug users who do not attend health and social services. The work of the outreach team involved educating these users in harm reduction and offering them health and social alternatives beyond the clinical trial

Improvement of Remote Sensing-Based Assessment of Defoliation of Pinus spp. Caused by Thaumetopoea pityocampa Denis and Schiffermüller and Related Environmental Drivers in Southeastern Spain

This study used Landsat temporal series to describe defoliation levels due to the Pine Processionary Moth (PPM) in Pinus forests of southeastern Andalusia (Spain), utilizing Google Earth Engine. A combination of remotely sensed data and field survey data was used to detect the defoliation levels of different Pinus spp. and the main environmental drivers of the defoliation due to the PPM. Four vegetation indexes were also calculated for remote sensing defoliation assessment, both inside the stand and in a 60-m buffer area. In the area of study, all Pinus species are affected by defoliation due to the PPM, with a cyclic behavior that has been increasing in frequency in recent years. Defoliation levels were practically equal for all species, with a high increase in defoliation levels 2 and 3 since 2014. The Moisture Stress Index (MSI) and Normalized Difference Infrared Index (NDII) exhibited similar overall (p < 0.001) accuracy in the assessment of defoliation due to the PPM. The synchronization of NDII-defoliation data had a similar pattern for all together and individual Pinus species, showing the ability of this index to adjust the model parameters based on the characteristics of specific defoliation levels. Using Landsat-based NDII-defoliation maps and interpolated environmental data, we have shown that the PPM defoliation in southeastern Spain is driven by the minimum temperature in February and the precipitation in June, March, September, and October. Therefore, the NDII-defoliation assessment seems to be a general index that can be applied to forests in other areas. The trends of NDII-defoliation related to environmental variables showed the importance of summer drought stress in the expansion of the PPM on Mediterranean Pinus species. Our results confirm the potential of Landsat time-series data in the assessment of PPM defoliation and the spatiotemporal patterns of the PPM; hence, these data are a powerful tool that can be used to develop a fully operational system for the monitoring of insect damage

Germination and Early Seedling Development in Quercus ilex Recalcitrant and Non-dormant Seeds: Targeted Transcriptional, Hormonal, and Sugar Analysis

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0–S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1–S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds.España Ministerio de Ciencia e Innovación AGL2009-12243-C02-0

Desarrollo de una metodología para la evaluación de proteínas secretadas por macrophomina phaseolina al medio de cultivo

Macrophomina phaseolina, es un hongo necrotrófico capaz de infectar cultivos de importancia económica, tales como la soja y el sésamo. Éste hongo secreta una amplia variedad de proteínas que le permiten invadir a la planta e iniciar el proceso de infección. Por lo tanto, numerosos estudios se basan en las proteínas con actividad hidrolílica secretadas por este hongo, sin embargo, las proteínas sin actividad enzimática también podrían estar implicadas en este proceso. En el presente trabajo se planteó el desarrollo de una metodología para la evaluación de proteínas secretadas por M. phaseolina al medio de cultivo Papa Dextrosa Caldo (PDB). Se optimizó una metodología de purificación de proteínas para la posterior evaluación del perfil proteico mediante una electroforesis unidimensional SDS-PAGE mediante la evaluación de tres metodologías de purificación de proteínas. El método más efectivo de purificación fue la precipitación de proteínas con ácido tricloroacético(TCA) el cual permitió obtener un rendimiento de 3,9 mg de proteína por gramos de liofilizado y un perfil proteico nítido para su análisisFil: Benegas López, Andrea C.. Universidad Nacional de Asunción (Paraguay)Fil: Flores-Giubi, M. Eugenia. Universidad Nacional de Asunción (Paraguay)Fil: Romero-Rodríguez, M. Cristina. Universidad Nacional de Asunción (Paraguay

Population Genetic Diversity of Quercus ilex subsp. ballota (Desf.) Samp. Reveals Divergence in Recent and Evolutionary Migration Rates in the Spanish Dehesas

The Spanish dehesas have been severely affected by human activities that date to the prehistoric period and have suffered accelerated decline since the 1980s. Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) is a key component of this system, and its acorns provide an important food source for wildlife and domesticated livestock. Our earlier work showed structured variation in acorn morphology and biochemistry. Here, we used chloroplast and nuclear microsatellites to detect genetic structure among populations of Q. ilex from the major biogeographic regions of Andalusia. We found high levels of spatial differentiation with chloroplast DNA indicating little seed dispersal among populations. Spatial differentiation was weaker for nuclear DNA, presumably as a result of more widespread pollen dispersal and its larger effective population size. The Baetic Cordillera (Cádiz) population consistently appeared well separated from populations of the northern Sierra Morena, suggesting that the Guadalquivir Valley has played an important role in determining population divergence. This may be, in part, evolutionary, as suggested by chloroplast DNA, and, in part, a result of human-induced population isolation, as Q. ilex has been removed from the Guadalquivir Valley. Evolutionary gene flow rates were greater than contemporary rates, which were limited to unidirectional gene flow from Córdoba to other populations in the Sierra Morena and, surprisingly, to the southern population at Almería. The inconsistency between evolutionary and recent migration rates suggests an effect of anthropogenic activity over the last few generations of Q. ilex

Performance characteristics of the whole-body discovery IQ PET/CT system

The aim of this study was to assess the physical performance of a new PET/CT system, the Discovery IQ with 5-ring detector blocks. Methods: Performance was measured using the National Electrical Manufacturers Association NU2-2012 methodology. Image quality was extended by accounting for different acquisition parameters (lesion-to-background ratios [8:1, 4:1, and 2:1] and acquisition times) and reconstruction algorithms (VUE-point HD [VPHD], VPHD with point-spread-function modeling [VPHD-S], and Q.Clear). Tomographic reconstruction was also assessed using a Jaszczak phantom. Additionally, 30 patient lesions were analyzed to account for differences in lesion volume and SUV quantification between reconstruction algorithms. Results: Spatial resolution ranged from 4.2 mm at 1 cm to 8.5 mm at 20 cm. Sensitivity measured at the center and at 10 cm was 22.8 and 20.4 kps/kBq, respectively. The noise-equivalent counting rate peak was 124 kcps at 9.1 kBq/cm3 The scatter fraction was 36.2%. The accuracy of correction for count losses and randoms was 3.9%. In the image quality test, contrast recovery for VPHD, VPHD-S, and Q.Clear ranged from 18%, 18%, and 13%, respectively (hot contrast; 10-mm sphere diameter; ratio, 2:1), to 68%, 67%, and 81%, respectively (cold contrast; 37-mm sphere diameter; ratio, 8:1). Background variability ranged from 3.4%, 3.0%, and 2.1%, respectively (ratio, 2:1), to 5.5%, 4.8%, and 3.7%, respectively (ratio, 8:1). On Q.Clear reconstruction, the decrease in the penalty term (β) increased the contrast recovery coefficients and background variability. With the Jaszczak phantom, image quality increased overall when a reconstruction algorithm modeling the point-spread function was used, and use of Q.Clear increased the signal-to-noise ratio. Lesions analyzed using VPHD-S and Q.Clear had an SUVmean of 6.5 ± 3 and 7 ± 3, respectively (P < 0.01), and an SUVmax of 11 ± 4.8 and 12 ± 4, respectively (P < 0.01). No significant difference in mean lesion volume was found between algorithms. Conclusion: Among the various Discovery bismuth germanium oxide-based PET/CT scanners, the IQ with 5-ring detector blocks has the highest overall performance, with improved sensitivity and counting rate performance. Q.Clear reconstruction improves the PET image quality, with higher recovery coefficients and lower background variability

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress