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Acknowledgments We would like to thank Jorge Dominguez and David Medel for preparation of plant material and fungal strains, and Elena Zafra for microsatellite amplification. We would also like to express our gratitude to the Spanish Elm Breeding and Conservation Programme for supporting this study. This research was funded by the Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA) and the Spanish National Research Plan (AGL2012-35580 and AGL2015-66925-R MINECO/FEDER, UE). P.P. acknowledges funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement PIEF-GA-2013-627761., 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, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, unpub

Acknowledgements The authors gratefully acknowledge the computer resources and technical support provided by the Plataforma Andaluza de Bioinformática of the University of Málaga, Spain. This study was supported by the Spanish Ministerio de Ciencia e Innovación [AGL2009-12139-C02-02, BIO2009-07490], the European Union [PLE2009-0016] and the Junta de Andalucía [CVI-6075 and BIO-114]., 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 deepe, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

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., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgments The authors would like to thank the three anonymous reviewers and the editor for their helpful comments and suggestions. This work has been funded through the projects AGL2006-03242/FOR (Spanish Ministry of Education and Science), CCG07-UPM/AMB-1932 and CCG10-UPM/AMB-5038 (Madrid Regional Government-UPM)., 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, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Elms (Ulmus spp.) were once dominant trees in mixed broadleaf forests of many European territories, mainly distributed near rivers and streams or on floodplains. Since ancient times they have provided important services to humans, and several selected genotypes have been massively propagated and planted. Today elm populations are severely degraded due to the negative impact of human-induced changes in riparian ecosystems and the emergence of the highly aggressive Dutch elm disease pathogens. Despite the death of most large elm specimens, there is no evidence of genetic diversity loss in elm populations, probably due to their ability to resprout after disease. The recovery of elm populations from the remaining diversity should build from genomic tools that facilitate achievement of resistant elm clones. Research works to date have discerned the genetic diversity of elms and are well on the way to deciphering the genetic clues of elm resistance and pathogen virulence, key findings for addressing recovery of elm populations. Several tolerant clones suitable for use in urban and landscape planting have been obtained through traditional species hybridization with Asian elms, and various native clones have been selected and used in pilot forest restoration projects. Successful reintroduction of elms should also rely on a deeper understanding of elm ecology, in particular their resilience to abiotic and biotic disturbances. However, all these efforts would be in vain without the final acceptance of elm reintroduction by the social actors involved, making it necessary to evaluate and publicize the ecosystem services elms can provide for today’s society., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Acknowledgments We would like to thank Jorge Dominguez for supplying the plant material and Juan Antonio Martín for supplying the fungal strains. This research was funded by the Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA), and by the Spanish National Research Plan (AGL2012-35580). We would also like to express our gratitude to the Spanish Elm Breeding and Conservation Programme., 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, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

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.

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.

One century after the first report of Dutch elm disease (DED), there is still no practical solution for this problem threatening European and American elms (Ulmus spp.). The long breeding cycles needed to select resistant genotypes and the lack of efficient treatments keep disease incidence at high levels. In this work, the expression of defense-related genes to the causal agent of DED, Ophiostoma novo-ulmi Brasier, was analyzed in in vitro clonal plantlets from two DED-resistant and two DED-susceptible Ulmus minor Mill. trees. In addition, the effect of the inoculation of an endophytic pink-pigmented yeast (Cystobasidium sp.) on the plant's defense system was tested both individually and in combination with O. novo-ulmi. The multifactorial nature of the resistance to DED was confirmed, as no common molecular response was found in the two resistant genotypes. However, the in vitro experimental system allowed discrimination of the susceptible from the resistant genotypes, showing higher levels of oxidative damage and phenolic compounds in the susceptible genotypes after pathogen inoculation. Inoculation of the endophyte before O. novo-ulmi attenuated the plant molecular response induced by the pathogen and moderated oxidative stress levels. Niche competition, endophyte-pathogen antagonism and molecular crosstalk between the host and the endophyte are discussed as possible mechanisms of stress reduction. In sum, our results confirm the complex and heterogeneous nature of DED resistance mechanisms and highlight the possibility of using certain endophytic yeasts as biological tools to improve tree resilience against biotic stress.

Pines are the dominant conifers in Mediterranean forests. As long-lived sessile organisms that seasonally have to cope with drought periods, they have developed a variety of adaptive responses. However, during last decades, highly intense and long-lasting drought events could have contributed to decay and mortality of the most susceptible trees. Among conifer species, Pinus pinaster Ait. shows remarkable ability to adapt to different environments. Previous molecular analysis of a full-sib family designed to study drought response led us to find active transcriptional activity of stress-responding genes even without water deprivation in tolerant genotypes. To improve our knowledge about communication between above- and below-ground organs of maritime pine, we have analyzed four graft-type constructions using two siblings as rootstocks and their progenitors, Gal 1056 and Oria 6, as scions. Transcriptomic profiles of needles from both scions were modified by the rootstock they were grafted on. However, the most significant differential gene expression was observed in drought-sensitive Gal 1056, while in drought-tolerant Oria 6, differential gene expression was very much lower. Furthermore, both scions grafted onto drought-tolerant rootstocks showed activation of genes involved in tolerance to abiotic stress, and is most remarkable in Oria 6 grafts where higher accumulation of transcripts involved in phytohormone action, transcriptional regulation, photosynthesis and signaling has been found. Additionally, processes, such as those related to secondary metabolism, were mainly associated with the scion genotype. This study provides pioneering information about rootstock effects on scion gene expression in conifers.

ACKNOWLEDGMENTS I. Aranda (INIA-CIFOR) is gratefully acknowledged for his valuable collaboration and support in obtaining and interpreting data on physiology. D. Sobral (Instituto Gulbenkian de Ciência, Portugal) is gratefully acknowledged for his advice in the bioinformatics analyses. Bartolomé Sabater is gratefully acknowledged for critical review of the manuscript. S. Ferrándiz (INIA-CIFOR) is gratefully acknowledged for helping with the preparation of the manuscript. This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2015-66048-C2-1-R; RTI2018-098015-B-I00; IMP2018-005)]; European Union's Seventh Framework Programme 2007-2013 (FP7-289841); EU H2020 Programme (H2020-676559); Fundação para a Ciência e a Tecnologia, FCT (IF/01168/2013; UID/Multi/04551/2013); and People Programme, Marie Curie Actions (PIEF-GA-2013-627761)., 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, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Funding This work has been funded by the research project AGL2015-66925-R (MINECO/FEDER) and by an agreement between Universidad Politécnica de Madrid and Dirección General de Desarrollo Rural y Política Forestal (MAPA/FEADER)., 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., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub