Core endophytic mycobiome in Ulmus minor and its relation to Dutch elm disease resistance.

The core microbiota of plants exerts key effects on plant performance and resilience to stress. The aim of this study was to identify the core endophytic mycobiome in U. minor stems and disentangle associations between its composition and the resistance to Dutch elm disease (DED). We also defined its spatial variation within the tree and among distant tree populations. Stem samples were taken i) from different heights of the crown of a 168-year-old elm tree, ii) from adult elm trees growing in a common garden and representing a gradient of resistance to DED, and iii) from trees growing in two distant natural populations, one of them with varying degrees of vitality. Endophyte composition was profiled by high throughput sequencing of the first internal transcribed spacer region (ITS1) of the ribosomal DNA. Three families of yeasts (Buckleyzymaceae, Trichomeriaceae and Bulleraceae) were associated to DED-resistant hosts. A small proportion (10%) of endophytic OTUs was almost ubiquitous throughout the crown while tree colonization by most fungal taxa followed stochastic patterns. A clear distinction in endophyte composition was found between geographical locations. By combining all surveys, we found evidence of a U. minor core mycobiome, pervasive within the tree and ubiquitous across locations, genotypes and health status.

Stem metabolism under drought stress - a paradox of increasing respiratory substrates and decreasing respiratory rates.

Metabolic changes underpinning drought-induced variations in stem respiration (Rs ) are unknown. We measured Rs rates and metabolite and gene expression profiles in Ulmus minor Mill. and Quercus ilex L. seedlings subjected to increasing levels of drought stress to better understand how carbon, nitrogen and energy metabolism interact during drought. In both species, only plants showing extreme stress symptoms - i.e. negligible rates of leaf stomatal conductance and photosynthesis, and high stem dehydration (30-50% of maximum water storage) and contraction (50-150 µm week-1 ) - exhibited lower Rs rates than well-watered plants. Abundance of low-molecular weight sugars (e.g. glucose and fructose) and sugar alcohols (e.g. mannitol) increased with drought, at more moderate stress and to a higher extent in Q. ilex than U. minor. Abundance of amino acids increased at more severe stress, more abruptly, and to a higher extent in U. minor, coinciding with leaf senescence, which did not occur in Q. ilex. Organic acids changed less in response to drought: threonate and glycerate increased, and citrate decreased although slightly in both species. Transcripts of genes coding for enzymes of the Krebs cycle decreased in Q. ilex and increased in U. minor in conditions of extreme drought stress. The maintenance of Rs under severe growth and photosynthetic restrictions reveals the importance of stem mitochondrial activity in drought acclimation. The eventual decline in Rs diverts carbon substrates from entering the Krebs cycle that may help to cope with osmotic and oxidative stress during severe drought and to recover hydraulic functionality afterwards.

Molecular study of drought response in the Mediterranean conifer Pinus pinaster Ait.: Differential transcriptomic profiling reveals constitutive water deficit-independent drought tolerance mechanisms.

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

Changes in plant function and root mycobiome caused by flood and drought in a riparian tree.

Under increasingly harsh climatic conditions, conservation of threatened species requires integrative studies to understand stress tolerance. Riparian Ulmus minor Mill. populations have been massively reduced by Dutch Elm disease (DED). However, resistant genotypes were selected to restore lost populations. To understand the acclimation mechanisms to the succession of abiotic stresses, ramets of five DED-tolerant U. minor genotypes were subjected to flood and subsequently to drought. Physiological and biochemical responses were evaluated together with shifts in root-fungal assemblages. During both stresses, plants exhibited a decline in leaf net photosynthesis and an increase in percentage loss of stem hydraulic conductivity and in leaf and root proline content. Stomatal closure was produced by chemical signals during flood and hydraulic signals during drought. Despite broad similarities in plant response to both stresses, root-mycobiome shifts were markedly different. The five genotypes were similarly tolerant to moderate drought, however, flood tolerance varied between genotypes. In general, flood did not enhance drought susceptibility due to fast flood recovery, nevertheless, different responses to drought after flood were observed between genotypes. Associations were found between some fungal taxonomic groups and plant functional traits varying with flood and drought (e.g. proline, chlorophyll and starch content) indicating that the thriving of certain taxa depends on host responses to abiotic stress.

Growth resilience and oxidative burst control as tolerance factors to Ophiostoma novo-ulmi in Ulmus minor.

The Dutch elm disease (DED) pathogens, Ophiostoma ulmi (Buisman) Nannf. and the more aggressive Ophiostoma novo-ulmi Brasier, have decimated European elm populations in the last 100 years. Today, the number of tolerant elm varieties available on the market is limited, partly due to the long breeding cycles and expensive facilities they require. Developing a low-cost technique to allow early screening of elm tolerance based on simple morphological and/or biochemical traits would considerably boost elm breeding and research. Within this general aim, we developed an in vitro plant culture system to (i) characterize stress responses to O. novo-ulmi-root inoculation in two Ulmus minor Mill. clones of contrasting susceptibility level to DED (termed 'tolerant' and 'susceptible') and (ii) compare the upward dispersal rate of the pathogen in the two clones. Constitutive xylem anatomy was similar in both clones, indicating that differences in plant responses to the pathogen are not attributable to anatomical factors (e.g., conduit size). Susceptible plantlets suffered a significant delay in apical growth and a decrease in chlorophyll content at 21 days post-inoculation (dpi). The rate of pathogen dispersal from roots to aerial tissues was similar in both clones. However, the tolerant clone showed a marked increase in lipid peroxidation at 1 dpi, while the susceptible clone showed enhanced values of lipid peroxidation during most of the experimental period (1-21 dpi). Despite wide stem colonization by the pathogen, the tolerant clone effectively regulated the oxidative stress levels and showed remarkable resilience to inoculation. These results extend current knowledge on elm defense mechanisms, and the proposed in vitro plant culture system emerges as a promising early screening method for tolerance to improve elm breeding.

A molecular approach to drought-induced reduction in leaf CO2 exchange in drought-resistant Quercus ilex.

Drought-induced reduction of leaf gas exchange entails a complex regulation of the plant leaf metabolism. We used a combined molecular and physiological approach to understand leaf photosynthetic and respiratory responses of 2-year-old Quercus ilex seedlings to drought. Mild drought stress resulted in glucose accumulation while net photosynthetic CO2 uptake (Pn ) remained unchanged, suggesting a role of glucose in stress signaling and/or osmoregulation. Simple sugars and sugar alcohols increased throughout moderate-to-very severe drought stress conditions, in parallel to a progressive decline in Pn and the quantum efficiency of photosystem II; by contrast, minor changes occurred in respiration rates until drought stress was very severe. At very severe drought stress, 2-oxoglutarate dehydrogenase complex gene expression significantly decreased, and the abundance of most amino acids dramatically increased, especially that of proline and γ-aminobutyric acid (GABA) suggesting enhanced protection against oxidative damage and a reorganization of the tricarboxylic cycle acid cycle via the GABA shunt. Altogether, our results point to Q. ilex drought tolerance being linked to signaling and osmoregulation by hexoses during early stages of drought stress, and enhanced protection against oxidative damage by polyols and amino acids under severe drought stress.

Gene expression trade-offs between defence and growth in English elm induced by Ophiostoma novo-ulmi.

Wilt diseases caused by vascular pathogens include some of the most damaging stresses affecting trees. Dutch elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, destroyed most of North American and European elm populations in the 20th century. The highly susceptible English elm, also known as Atinian clone, suffered the highest mortality rates during the last pandemic event, probably due to its lack of genetic diversity. To study the DED pathosystem, we inoculated English elm ramets with O. novo-ulmi and evaluated xylem anatomy, molecular response, and disease symptoms. The high DED susceptibility of the clone was linked to xylem structure. The transcript levels changed significantly for 1,696 genes during O. novo-ulmi invasion. Genes covering different steps of the plant immune system were identified, many of which showed homology with Arabidopsis thaliana genes involved in systemic acquired resistance. Induction of several pathogenesis-related proteins and repression of fasciclin-like arabinogalactan proteins and other cell wall biosynthesis pathways evidence unbalanced costs between growth and defence mechanisms far from the inoculation point. This study sheds light on elm molecular defence mechanisms against DED.

Massive sequencing of Ulmus minor's transcriptome provides new molecular tools for a genus under the constant threat of Dutch elm disease.

Elms, especially Ulmus minor and U. americana, are carrying out a hard battle against Dutch elm disease (DED). This vascular wilt disease, caused by Ophiostoma ulmi and O. novo-ulmi, appeared in the twentieth century and killed millions of elms across North America and Europe. Elm breeding and conservation programmes have identified a reduced number of DED tolerant genotypes. In this study, three U. minor genotypes with contrasted levels of tolerance to DED were exposed to several biotic and abiotic stresses in order to (i) obtain a de novo assembled transcriptome of U. minor using 454 pyrosequencing, (ii) perform a functional annotation of the assembled transcriptome, (iii) identify genes potentially involved in the molecular response to environmental stress, and (iv) develop gene-based markers to support breeding programmes. A total of 58,429 putative unigenes were identified after assembly and filtering of the transcriptome. 32,152 of these unigenes showed homology with proteins identified in the genome from the most common plant model species. Well-known family proteins and transcription factors involved in abiotic, biotic or both stresses were identified after functional annotation. A total of 30,693 polymorphisms were identified in 7,125 isotigs, a large number of them corresponding to single nucleotide polymorphisms (SNPs; 27,359). In a subset randomly selected for validation, 87% of the SNPs were confirmed. The material generated may be valuable for future Ulmus gene expression, population genomics and association genetics studies, especially taking into account the scarce molecular information available for this genus and the great impact that DED has on elm populations.

Nucleotide polymorphisms in a pine ortholog of the Arabidopsis degrading enzyme cellulase KORRIGAN are associated with early growth performance in Pinus pinaster.

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 korrigan SNPs by ridge regression-best linear unbiased prediction (RR-BLUP) and cross-validation accounted for up to 8 and 15% of the phenotypic variance for height and polycyclic growth, respectively, and did not improve adding SNPs from other growth-related candidate genes. For root-collar diameter and total biomass, they accounted for 1.6 and 1.1% of the phenotypic variance, respectively, but increased to 15 and 4.1% when other SNPs from lp3.1, lp3.3 and cad were included in RR-BLUP models. These results point towards a desirable integration of candidate-gene studies as a means to pre-select relevant markers, and aid genomic selection in maritime pine breeding programs.

Proliferation of axial parenchymatic xylem cells is a key step in wound closure of girdled stems in Pinus canariensis.

BACKGROUND: Wounds caused by fire, herbivorism, rock impacts, etc. cause the direct loss of photosynthetic, storage and/or vascular tissue. In addition, they may entail other damages, such as desiccation of the exposed internal parts, or become a gateway to infection by fungi and other pathogens. To successfully overcome such injuries, plants must reorganize their meristems or even differentiate new ones, producing new traumatic tissues to cover the wound and restore the vascular connection. RESULTS: In this work we analyse the anatomical growth response in conifers after debarking and injuring the vascular cambium, using Pinus canariensis as model species, due to its high wound recovery ability. Conversely to angiosperm woody species, this process is initiated and largely driven by the damaged vascular cambium and not by proliferation in the wound surface. We have detected alterations and switches in the divisions of cambial cells, associated to their position relative to the surface and edges of the wound, resulting in disordered traumatic xylem. We also describe the formation of column-like structures, after girdling, which are in part formed by the proliferation of xylem parenchymatous cells, associated to axial resin ducts. CONCLUSIONS: Abundant resinosis on the wound surface, typical of conifers, is an efficient barrier against opportunistic fungi, insects, etc. but it also hinders the healing process directly from the surface. Thus, wound closure must be largely carried out from the wound margins, being a much slower process, which very often remains unconcluded for long years. This work also describes for the first time the proliferation of inner parenchymatous cells to form column-like structures, which accelerates wound closure in girdled P. canariensis. Irregularities in the surface of the healing edge or column-like structures result in the production of disordered vascular tissues, compromising their future functionality, and which must be overcome through the fast restoration of the proper polarity in vascular cambium.

Novel dehydrins lacking complete K-segments in Pinaceae. The exception rather than the rule.

Dehydrins are thought to play an essential role in the plant response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins contain conserved and repeated segments in their amino acid sequence, used for their classification. Thus, dehydrins from angiosperms present different repetitions of the segments Y, S, and K, while gymnosperm dehydrins show A, E, S, and K segments. The only fragment present in all the dehydrins described to date is the K-segment. Different works suggest the K-segment is involved in key protective functions during dehydration stress, mainly stabilizing membranes. In this work, we describe for the first time two Pinus pinaster proteins with truncated K-segments and a third one completely lacking K-segments, but whose sequence homology leads us to consider them still as dehydrins. qRT-PCR expression analysis show a significant induction of these dehydrins during a severe and prolonged drought stress. By in silico analysis we confirmed the presence of these dehydrins in other Pinaceae species, breaking the convention regarding the compulsory presence of K-segments in these proteins. The way of action of these unusual dehydrins remains unrevealed.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification.

BACKGROUND: Understanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought. RESULTS: High density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area. CONCLUSIONS: The integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.

Vulnerability to cavitation, hydraulic efficiency, growth and survival in an insular pine (Pinus canariensis).

BACKGROUND AND AIMS: It is widely accepted that hydraulic failure due to xylem embolism is a key factor contributing to drought-induced mortality in trees. In the present study, an attempt is made to disentangle phenotypic plasticity from genetic variation in hydraulic traits across the entire distribution area of a tree species to detect adaptation to local environments. METHODS: A series of traits related to hydraulics (vulnerability to cavitation and hydraulic conductivity in branches), growth performance and leaf mass per area were assessed in eight Pinus canariensis populations growing in two common gardens under contrasting environments. In addition, the neutral genetic variability (FST) and the genetic differentiation of phenotypic variation (QST) were compared in order to identify the evolutionary forces acting on these traits. KEY RESULTS: The variability for hydraulic traits was largely due to phenotypic plasticity. Nevertheless, the vulnerability to cavitation displayed a significant genetic variability (approx. 5 % of the explained variation), and a significant genetic × environment interaction (between 5 and 19 % of the explained variation). The strong correlation between vulnerability to cavitation and survival in the xeric common garden (r = -0·81; P < 0·05) suggests a role for the former in the adaptation to xeric environments. Populations from drier sites and higher temperature seasonality were less vulnerable to cavitation than those growing at mesic sites. No trade-off between xylem safety and efficiency was detected. QST of parameters of the vulnerability curve (0·365 for P50 and the slope of the vulnerability curve and 0·452 for P88) differed substantially from FST (0·091), indicating divergent selection. In contrast, genetic drift alone was found to be sufficient to explain patterns of differentiation for xylem efficiency and growth. CONCLUSIONS: The ability of P. canariensis to inhabit a wide range of ecosystems seemed to be associated with high phenotypic plasticity and some degree of local adaptations of xylem and leaf traits. Resistance to cavitation conferred adaptive potential for this species to adapt successfully to xeric conditions.

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.

Annotated genetic linkage maps of Pinus pinaster Ait. from a Central Spain population using microsatellite and gene based markers.

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 linkage maps reporting QTL information revealed 11 annotated SNPs in candidate genes that co-localized with previously reported QTLs for wood properties and water use efficiency. CONCLUSIONS: This study provides genetic linkage maps from a Spanish population that shows high levels of genetic divergence with French populations from which segregating progenies have been previously mapped. These genetic maps will be of interest to construct a reliable consensus linkage map for the species. The importance of developing functional genetic linkage maps is highlighted, especially when working with breeding populations for its future application in MAS for traits of interest.

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.

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

BACKGROUND: Pinus 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. DESCRIPTION: EuroPineDB 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. CONCLUSIONS: The 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.

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.