Álvaro Soto

GENETICALLY DEPAUPERATE BUT WIDESPREAD: THE CASE OF AN EMBLEMATIC MEDITERRANEAN PINE

Abstract Genetic variation is generally considered a prerequisite for adaptation to new environmental conditions. Thus the discovery of genetically depauperate but geographically widespread species is unexpected. We used 12 paternally inherited chloroplast microsatellites to estimate population genetic variation across the full range of an emblematic circum-Mediterranean conifer, stone pine (Pinus pinea L.). The same chloroplast DNA haplotype is fixed in nearly all of the 34 investigated populations. Such a low level of variation is consistent with a previous report of very low levels of diversity at nuclear loci in this species. Stone pine appears to have passed through a severe and prolonged demographic bottleneck, followed by subsequent natural- and human-mediated dispersal across the Mediterranean Basin. No other abundant and widespread plant species has as little genetic diversity as P. pinea at both chloroplast and nuclear markers. However, the species harbors a nonnegligible amount of variation at adaptive traits. Thus a causal relationship between genetic diversity, as measured by marker loci, and the evolutionary precariousness of a species, cannot be taken for granted.

Phylogeography: English elm is a 2,000-year-old Roman clone

The outbreak of Dutch elm disease in the 1970s ravaged European elm populations, killing more than 25 million trees in Britain alone; the greatest impact was on Ulmus procera, otherwise known as the English elm. Here we use molecular and historical information to show that this elm derives from a single clone that the Romans transported from Italy to the Iberian peninsula, and from there to Britain, for the purpose of supporting and training vines. Its highly efficient vegetative reproduction and its inability to set seeds have preserved this clone unaltered for 2,000 years as the core of the English elm population — and the preponderance of this susceptible variety may have favoured a rapid spread of the disease.

Elucidating the role of genetic drift and natural selection in cork oak differentiation regarding drought tolerance

Drought is the main selection agent in Mediterranean ecosystems and it has been suggested as an important evolutionary force responsible for population diversification in these types of environments. However, population divergence in quantitative traits can be driven by either natural selection, genetic drift or both. To investigate the roles of these forces on among‐population divergence in ecophysiological traits related to drought tolerance (carbon isotope discrimination, specific leaf area, leaf size and leaf nitrogen content), we compared molecular and quantitative genetic differentiation in a common garden experiment including thirteen cork oak (Quercus suber L.) populations across a gradient of rainfall and temperature. Population differentiation for height, specific leaf area, leaf size and nitrogen leaf content measured during a dry year far exceeded the molecular differentiation measured by six nuclear microsatellites. Populations from dry‐cool sites showed the lowest nitrogen leaf content and the smallest and thickest leaves contrasting with those from humid‐warm sites. These results suggest (i) these traits are subjected to divergence selection and (ii) the genetic differences among populations are partly due to climate adaptation. By contrast, the low among‐population divergence found in basal diameter, annual growth and carbon isotopic discrimination (a surrogate for water use efficiency) suggests low or no divergence selection for these traits. Among‐population differentiation for neutral markers was not a good predictor for differentiation regarding the quantitative traits studied here, except for leaf size. The correlation observed between the genetic differentiation for leaf size and that for molecular markers was exclusively due to the association between leaf size and the microsatellite QpZAG46, which suggests a possible linkage between QpZAG46 and genes encoding for leaf size.

Protein Cryoprotective Activity of a Cytosolic Small Heat Shock Protein That Accumulates Constitutively in Chestnut Stems and Is Up-Regulated by Low and High Temperatures

Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long‐term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species‐specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold‐enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold‐tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.

EuroPineDB: a high-coverage web database for maritime pine transcriptome

BackgroundPinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases.DescriptionEuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided.ConclusionsThe EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Novel conserved segments are associated with differential expression patterns for Pinaceae dehydrins

Dehydrins are thought to play an essential role in the response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins have been classified into five groups according to the presence of conserved and repeated motifs in their amino acid sequence. Due to their putative functions in the response to stress, dehydrins have been often used as candidate genes in studies on population variability and local adaptation to environmental conditions. However, little is still known regarding the differential role played by such groups or the mechanism underlying their function. Based on the sequences corresponding to dehydrins available in public databases we have isolated eight different dehydrins from cDNA of Pinus pinaster. We have obtained also their genomic sequences and identified their intron/exon structure. Quantitative RT-PCR analysis of their expression pattern in needles, stems and roots during a severe and prolonged drought stress, similar to the ones trees must face in nature, is also reported. Additionally, we have identified two amino acid motifs highly conserved and repeated in Pinaceae dehydrins and absent in angiosperms, presumably related to the divergent expression profiles observed.

Identification of water stress genes in Pinus pinaster Ait. by controlled progressive stress and suppression-subtractive hybridization

Climate change is a major challenge particularly for forest tree species, which will have to face the severe alterations of environmental conditions with their current genetic pool. Thus, an understanding of their adaptive responses is of the utmost interest. In this work we have selected Pinus pinaster as a model species. This pine is one of the most important conifers (for which molecular tools and knowledge are far more scarce than for angiosperms) in the Mediterranean Basin, which is characterised in all foreseen scenarios as one of the regions most drastically affected by climate change, mainly because of increasing temperature and, particularly, by increasing drought. We have induced a controlled, increasing water stress by adding PEG to a hydroponic culture. We have generated a subtractive library, with the aim of identifying the genes induced by this stress and have searched for the most reliable expressional candidate genes, based on their overexpression during water stress, as revealed by microarray analysis and confirmed by RT-PCR. We have selected a set of 67 candidate genes belonging to different functional groups that will be useful molecular tools for further studies on drought stress responses, adaptation, and population genomics in conifers, as well as in breeding programs.

Molecular response to water stress in two contrasting Mediterranean pines (Pinus pinaster and Pinus pinea).

Adaptation to water stress has determined the evolution and diversification of vascular plants. Water stress is forecasted to increase drastically in the next decades in certain regions, such as in the Mediterranean basin. Consequently, a proper knowledge of the response and adaptations to drought stress is essential for the correct management of plant genetic resources. However, most of the advances in the understanding of the molecular response to water stress have been attained in angiosperms, and are not always applicable to gymnosperms. In this work we analyse the transcriptional response of two emblematic Mediterranean pines, Pinus pinaster and Pinus pinea, which show noticeable differences in their performance under water stress. Using microarray analysis, up to 113 genes have been detected as significantly induced by drought in both species. Reliability of expression patterns has been confirmed by RT-PCR. While induced genes with similar profiles in both species can be considered as general candidate genes for the study of drought response in conifers, genes with diverging expression patterns can underpin the differences displayed by these species under water stress. Most promising candidate genes for drought stress response include genes related to carbohydrate metabolism, such as glycosyltransferases or galactosidases, sugar transporters, dehydrins and transcription factors. Additionally, differences in the molecular response to drought and polyethylene-glycol-induced water stress are also discussed.

Drought Response in Forest Trees: From the Species to the Gene

Forest tree species, considering their long lifespan, symbolize one of the best biological examples of adaptation to a frequently changing harsh terrestrial environment. The adaptation to environments with water scarcity was the first challenge in the evolution of terrestrial photosynthetic organisms, and prompted the development of strategies and mechanisms to cope with drought. In this respect, the particular evolution and life history of forest tree species have brought about a plethora of specific adaptations to dry environments. The presence of a hydraulic system for long distance water transport and the need of maintaining functional tissues and organs for long periods of time are two important characteristics making forest tree species singular organisms within the plant kingdom. Selective pressure has prompted a variety of strategies in the control of water losses to maintain the functionality of the hydraulic system without compromising the carbon balance of the plant. These and other physiological responses focussed to increase the dehydration tolerance of tissues (e.g., osmotic adjustment) have played an important role in the development of specific adaptations under water limiting conditions. The adaptive changes are observable at different scales: from the population to the species, from the individual to the gene. The advance of high-throughput technologies will enable to unveil the complex interplay between phenotype and genotype. Genomic, proteomic, transcriptomic, and metabolomic approaches are beginning to bring light to the molecular basis of adaptation to drought in forest tree species. These new technologies, combined with more traditional approaches, will improve our current knowledge of the functional and molecular basis underlying adaptation and evolution of forest tree species living under dry environments. In this respect, this chapter covers some aspects of adaptation to drought at different integrative levels, from an ecophysiological perspective to a molecular-based point of view.