Nuria de María

The adaptive capacity of a species and its population is determined by both genetic and epigenetic variation, which defines the potential for adaptive evolution and plastic response to environmental changes. In this study, we used Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism (AFLP), similar genome-wide profiling techniques, to analyze the epigenetic and genetic variability in European beech provenances from Germany (DE), Spain (ES) and Sweden (SE), representing the latitudinal distribution of the species. In addition, we evaluated the effect of moderate water stress on cytosine methylation dynamics by comparing two latitudinal contrasting provenances. Our analysis revealed that trees from ES showed lower values of epigenetic and genetic diversity than those from DE and SE. Analyses of molecular variance for MSAPs and AFLPs showed that 16% and 15% of the among population variations were associated with epigenetic and genetic variation...

Trabajo presentado en la X Reunión Nacional de Fijación de Nitrógeno, celebrada en Granada (España), del 15 al 18 de junio de 2004El glifosato es uno de los herbicidas más empleados actualmente en cultivos agrícolas, por su inmovilidad, rápida inactivación y degradación en el suelo (2). En trabajos anteriores (1), hemos comprobado que este herbicida produce un rápido descenso de la actividad fijadora de nitrógeno así como ligeras alteraciones en el patrón polipeptídico de hojas y nódulos de plantas de Lupinus albus cv. Multolupa, inoculadas con Bradyrhizobium sp. (Lupinus) cepa ISLU-16. Sin embargo, la mayor alteración encontrada fue la presencia de un polipéptido de 44 kDa (44K) en el citosol bacteroidal, que no se detectaba en el citosol de bacteroides de plantas control. Se ha llevado a cabo la identificación de esta proteína mediante la secuenciación de su región N-terminal, la caracterización de sus genes codificadores, el análisis del patrón de expresión de su mRNA y el análisis de su estructura. El análisis de la secuencia nucleotídica de su gen codificador y de las secuencias aminoacídicas deducidas, mostraron que el polipéptido 44K posee una elevada homología de secuencia con porinas de la membrana externa de otras bacterias Gram negativas. Los ensayos de Southern del DNA genómico de la cepa ISLU-16 son indicativos de que esta proteína está codificada por un único gen en el genoma bacteriano. El patrón de expresión del gen mediante Northern de los RNA aislados de nódulos tratados con distintas dosis de herbicida, reveló una expresión constitutiva basal que se incrementa en los tratamientos con altas dosis de herbicida (5 y 10 mM), indicando que la expresión del polipéptido 44K está regulada transcripcionalmente. En la bacteria en vida libre se detectó sólo acumulación de RNA, cuando fue crecida en ausencia de glifosato, indicando diferencias en su patrón de expresión en vida libre y en simbiosis. Finalmente, hemos analizado la estructura de la proteína 44K. Se han identificado dos motivos: uno de asociación a membrana, próximo a la región C-t y otro característico de la subfamilia de porinas, situado a 30 aa de la región N-t. La estructura secundaria contiene esencialmente lámina β y un posible dominio transmembrana β-barrel correspondiente a la cadena P de una porina específica de sacarosa, presente en proteínas de membrana externa de enterobacterias. Actualmente se están estudiando la(s) posible(s) función(es) de esta proteína en la respuesta del microsimbionte frente al tratamiento con herbicidas

Trabajo presentado en la X Reunión Nacional de Fijación de Nitrógeno, celebrada en Granada (España), del 15 al 18 de junio de 2004El glifosato es uno de los herbicidas más empleados actualmente en cultivos agrícolas, por su inmovilidad, rápida inactivación y degradación en el suelo (2). En trabajos anteriores (1), hemos comprobado que este herbicida produce un rápido descenso de la actividad fijadora de nitrógeno así como ligeras alteraciones en el patrón polipeptídico de hojas y nódulos de plantas de Lupinus albus cv. Multolupa, inoculadas con Bradyrhizobium sp. (Lupinus) cepa ISLU-16. Sin embargo, la mayor alteración encontrada fue la presencia de un polipéptido de 44 kDa (44K) en el citosol bacteroidal, que no se detectaba en el citosol de bacteroides de plantas control. Se ha llevado a cabo la identificación de esta proteína mediante la secuenciación de su región N-terminal, la caracterización de sus genes codificadores, el análisis del patrón de expresión de su mRNA y el anális...

Increasing temperatures along with severe droughts are factors that may jeopardize the survival of the forests in the Mediterranean basin. In this region, Pinus pinaster is a common conifer species, that has been used as a model species in evolutionary studies due to its adaptive response to changing environments. Although its drought tolerance mechanisms are already known, knowledge about the dynamics of its root microbiota is still scarce. We aimed to decipher the structural (bacterial abundance), compositional, functional and associative changes of the P. pinaster rhizosphere bacterial communities in spring and summer, at DNA and RNA level (environmental DNA, live and dead cells, and those synthesizing proteins). A fundamental aspect of root microbiome-based approaches is to guarantee the correct origin of the samples. Thus, we assessed the genotype of host needles and roots from which rhizosphere samples were obtained. For more than 50% of the selected trees, genotype discrepancies were found and in three cases the plant species could not be determined. Rhizosphere bacterial communities were homogeneous with respect to diversity and structural levels regardless of the host genotype in both seasons. Nonetheless, significant changes were seen in the taxonomic profiles depending on the season. Seasonal changes were also evident in the bacterial co-occurrence patterns, both in DNA and RNA libraries. While spring communities switched to more complex networks, summer populations resulted in more compartmentalized networks, suggesting that these communities were facing a disturbance. These results may mirror the future status of bacterial communities in a context of climate change. A keystone hub was ascribed to the genus Phenylobacterium in the functional network calculated for summer. Overall, it is important to validate the origin and identity of plant samples in any plant-microbiota study so that more reliable ecological analyses are performed.

The PpDR transcriptome joins information from previous <i>Pinus pinaster</i> transcriptome together with 454 sequencing of 12 libraries from different organs from plants subjected to well watered or water stress regime. Sequencing yielded a total of 2,416,362 reads, which were mapped against the ProCoGen reference transcriptome containing 191,544 transcripts. Unmapped reads were used for <i>de novo </i>assembly, allowing the identification of 8,700 newly assembled transcripts longer than 200bp, which were added to the reference transcriptome to obtain the "<i>Pinus pinaster </i>drought response transcriptome" (PpDR transcriptome).<br>Files; Sequences in fasta format, Transcripts information table, Transcriptome annotation and Blast2GO file.<br>Departamento de Ecología y Genética Forestal, Centro de Investigación Forestal (CIFOR), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, ES.<br&...

We have carried out a candidate-gene-based association genetic study in Pinus pinaster Aiton and evaluated the predictive performance for genetic merit gain of the most significantly associated genes and single nucleotide polymorphisms (SNPs). We used a second generation 384-SNP array enriched with candidate genes for growth and wood properties to genotype mother trees collected in 20 natural populations covering most of the European distribution of the species. Phenotypic data for total height, polycyclism, root-collar diameter and biomass were obtained from a replicated provenance-progeny trial located in two sites with contrasting environments (Atlantic vs Mediterranean climate). General linear models identified strong associations between growth traits (total height and polycyclism) and four SNPs from the korrigan candidate gene, after multiple testing corrections using false discovery rate. The combined genomic breeding value predictions assessed for the four associated korriga...

Background Pinus pinaster Ait. is a major resin producing species in Spain. Genetic linkage mapping can facilitate marker-assisted selection (MAS) through the identification of Quantitative Trait Loci and selection of allelic variants of interest in breeding populations. In this study, we report annotated genetic linkage maps for two individuals (C14 and C15) belonging to a breeding program aiming to increase resin production. We use different types of DNA markers, including last-generation molecular markers. Results We obtained 13 and 14 linkage groups for C14 and C15 maps, respectively. A total of 211 and 215 markers were positioned on each map and estimated genome length was between 1,870 and 2,166 cM respectively, which represents near 65% of genome coverage. Comparative mapping with previously developed genetic linkage maps for P. pinaster based on about 60 common markers enabled aligning linkage groups to this reference map. The comparison of our annotated linkage maps and lin...

While recent advances have been gained on genome evolution in angiosperm lineages, virtually nothing is known about karyotype evolution in the other group of seed plants, the gymnosperms. Here we used high density gene-based linkage mapping to compare the karyotype structure of two families of conifers (the most abundant group of gymnosperms) separated around 290 million years ago: Pinaceae and Cupressaceae. We propose for the first time a model based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of Pinaceae (with n=12) and Cupressaceae (with n=11). The considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the Pinaceae. It also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes.

An increasing number of miRNAs and miRNA-related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well-watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA-Like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24-nt novel miRNA stood out, were strongly down-regulated. Given the putative roles of the miRNA-targeted transcripts validated specifically in root tissues, som...

Drought is a major environmental stress factor that determines the growth, development and survival of plants living in water scarce environments. Climate change predictions point at increasing dryness over the Mediterranean region. Pinus pinaster Ait. is an important Mediterranean conifer subjected to recurrent drought periods. Notwithstanding its relatively small geographical range this species, which is found along a rainfall cline, is characterized by a significant genetic and adaptive diversity. Different morphological and physiological responses appear to play an important role in drought adaptation of this conifer. These complex responses to drought, from perception to transcriptional, metabolic and physiological changes, need to be considered at a global systems biology level to study the multiple interactive components. Integration and analysis of multidisciplinary datasets will likely increase our understanding of molecular mechanisms controlling Pinus pinaster response to...

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Drought is an important driver of plant survival, growth, and distribution. Water deficit affects different pathways of metabolism, depending on plant organ. While previous studies have mainly focused on the metabolic drought response of a single organ, analysis of metabolic differences between organs is essential to achieve an integrated understanding of the whole plant response. In this work, untargeted metabolic profiling was used to examine the response of roots, stems, adult and juvenile needles from Pinus pinaster Ait. full-sib individuals, subjected to a moderate and long lasting drought period. Cyclitols content showed a significant alteration, in response to drought in all organs examined, but other metabolites increased or decreased differentially depending on the analyzed organ. While a high number of flavonoids were only detected in aerial organs, an induction of the glutathione pathway was mainly detected in roots. This result may reflect different antioxidant mechanisms activated in aerial organs and roots. Metabolic changes were more remarkable in roots than in the other organs, highlighting its prominent role in the response to water stress. Significant changes in flavonoids and ascorbate metabolism were also observed between adult and juvenile needles, consistent with previously proven differential functional responses between the two developmental stages. Genetic polymorphisms in candidate genes coding for a Myb1 transcription factor and a malate dehydrogenase (EC 1.1.1.37) were associated with different concentration of phenylalanine, phenylpropanoids and malate, respectively. The results obtained will support further research on metabolites and genes potentially involved in functional mechanisms related to drought tolerance in trees.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

The effects of glyphosate on protein metabolism, mesophyll cell ultrastructure and nodule ultrastructure and functioning of Lupinus albus cv. Multolupa inoculated with Bradyrhizobium sp. (Lupinus) were investigated. Young leaves and nodules were especially affected because these organs act as sinks of the herbicide. The alterations on nodular and chloroplast ultrastructure varied depending on herbicide concentration and time of exposure. After 3 days of 2.5 mM glyphosate application some toxic effects were detected. The most important alterations on nodules were the progressive cellular degradation of plant and bacteroidal cytosol and the rupture of bacteroidal membrane, whilst the peribacteroid membrane of the symbiosomes was preserved. This is the first report on the effect of glyphosate on legume-nodule ultrastructure. Glyphosate inhibited B. sp. (Lupinus) growth at concentrations higher than 62.5 microM. In the mesophyll cells, gradual disorganization of grana and intergrana was observed, loosing the parallel alignment with the chloroplast axis. As in nodules, degradation of membrane systems was observed, with the deformation, and even the rupture, of the tonoplast. These progressive effects were similar to those described in senescence processes. The adverse effects produced on infected zone can be due both to a direct effect of the herbicide on microsymbiont and to an indirect effect of glyphosate action on photosynthetic apparatus. Glyphosate produced changes in nodule cytosol and bacteroid proteins content and polypeptide pattern of leaves and nodules. With respect to proteins related to the oxygen diffusion mechanism, a large decrease in leghemoglobin and glycoproteins (recognized by antibodies MAC236 and MAC265) content was detected, which suggests that the oxygen diffusion mechanisms were also affected by glyphosate.