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Ectomycorrhizas have been reported to increase plant tolerance to drought. However, the mechanisms involved are not yet fully understood. Membranes are the first targets of degradation during drought, and growing evidences support a role for membrane lipids in plant tolerance and adaptation to drought. We have previously shown that improved tolerance of ectomycorrhizal oak plants to drought could be related to leaf membrane lipid metabolism, namely through an increased ability to sustain fatty acid content and composition, indicative of a higher membrane stability under stress. Here, we analysed in deeper detail the modulation of leaf lipid metabolism in oak plants mycorrhized with Pisolithus tinctorius and subjected to drought stress. Results show that mycorrhizal plants show patterns associated with water deficit tolerance, like a higher content of chloroplast lipids, whose levels are maintained upon drought stress. Likewise, mycorrhizal plants show increased levels of unsaturated fatty acids in the chloroplast phosphatidylglycerol lipid fraction. As a common response to drought, the digalactosyldiacyloglycerol/monogalactosyldiacyloglycerol ratio increased in the non-mycorrhizal plants, but not in the mycorrhizal plants, associated to smaller alterations in the expression of galactolipid metabolism genes, indicative of a higher drought tolerance. Under drought, inoculated plants showed increased expression of genes involved in neutral lipids biosynthesis, which could be related to an increased ability to tolerate drought stress. Overall, results from this study provide evidences of the involvement of lipid metabolism in the response of ectomycorrhizal plants to water deficit and point to an increased ability to maintain a stable chloroplast membrane functional integrity under stress.

Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598- 021- 87886-5 Mycorrhizas are known to have a positive impact on plant growth and ability to resist major biotic and abiotic stresses. However, the metabolic alterations underlying mycorrhizal symbiosis are still understudied. By using metabolomics and transcriptomics approaches, cork oak roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius were compared with non-colonized roots. Results show that compounds putatively corresponding to carbohydrates, organic acids, tannins, longchain fatty acids and monoacylglycerols, were depleted in ectomycorrhizal cork oak colonized roots. Conversely, non-proteogenic amino acids, such as gamma-aminobutyric acid (GABA), and several putative defense-related compounds, including oxylipin-family compounds, terpenoids and B6 vitamers were induced in mycorrhizal roots. Transcriptomic analysis suggests the involvement of GABA in ectomycorrhizal symbiosis through increased synthesis and inhibition of degradation in mycorrhizal roots. Results from this global metabolomics analysis suggest decreases in root metabolites which are common components of exudates, and in compounds related to root external protective layers which could facilitate plant-fungal contact and enhance symbiosis. Root metabolic pathways involved in defense against stress were induced in ectomycorrhizal roots that could be involved in a plant mechanism to avoid uncontrolled growth of the fungal symbiont in the root apoplast. Several of the identified symbiosis-specific metabolites, such as GABA, may help to understand how ectomycorrhizal fungi such as P. tinctorius benefit their host plants info:eu-repo/semantics/publishedVersion

An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest 32 species is needed to optimize forest sustainable productivity and thus to improve forests 33 services and their capacity to act as carbon sinks. In this study we investigated the 34 response of an oak species to ectomycorrhizae formation using a proteomics approach 35 complemented by biochemical analysis of carbohydrates levels. Comparative proteome 36 analysis between mycorrhizal and non-mycorrhizal cork oak plants revealed no 37 differences at the foliar level. However, the protein profile of 34 unique oak proteins 38 was altered in the roots. Consistent with the results of the biochemical analysis, the 39 proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose 40 for the metabolic activity of mycorrhizal roots which is consistent with an increased 41 allocation of carbohydrates from the plant to the fungus in order to sustain the 42 symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of 43 defense reactions, increased nutrient mobilization from the plant-fungus interface (N 44 and P), as well as cytoskeleton rearrangements and induction of plant cell wall 45 loosening for fungal root accommodation in colonized roots, are also suggested by the 46 results. The suggested improvement in root capacity to take up nutrients accompanied 47 by an increase of root biomass without apparent changes in aboveground biomass 48 strongly re-enforce the potential of mycorrizal inoculation to improve cork oak forest 49 resistance capacity to cope with coming climate change.

This study aims at identifying mammary gland genes expressed in Brazilian Holstein cattle produced under tropical conditions, as compared to the Portuguese Holstein cattle produced in a temperate region. For this purpose, cDNA microarrays and real-time (RT) PCR transcriptomic techniques were utilized in 12 Holstein cows from the same lactating phase and management systems divided into two groups: Holstein Brazil (HB) originated from Brazil and Holstein Portugal (HP) from Portugal. The genomic results show that from a total of 4608 genes available from the microarray slide (Bovine Long Oligo (BLO) library), 65 transcripts were identified as differentially expressed in mammary glands. The genes associated with mammary gland development and heat stress responses showed greater expression in HB animals. In the HP group, upregulated genes related with apoptosis and vascular development and downregulated genes related with resistance to heat stress were observed. Validation of microarray results was done using RT-PCR. HB animals had higher blood levels of growth hormone than HP animals. Blood levels of prolactin and T3 were similar for both groups and GH levels were increased in the HB group. The results suggest a gene change towards long-term acclimatization of Brazilian Holstein cattle to cope with tropical heat stress conditions.

Pisolithus tinctorius (Pers.) Coker and Couch (Syn.p P. arhizus [Scop.: Pers.] Rauschert) is an ectomycorrhizal fungus that interacts with some of the most important tree genera from temperate forests. In this chapter, we focus on the current knowledge about the beneficial role of P. tinctorius on its host plants. P. tinctorius has an overall positive effect on plant performance, which is often translated into increased shoot growth (height and weight), higher photosynthetic capacity, and mineral nutrient acquisition. Under environmental stress conditions, such as water deficit or soil chemical contamination, plant inoculation with P. tinctorius can afford some degree of tolerance, with mycorrhizal plants showing lower growth decline and less stress symptoms. However, the mechanisms involved in this tolerance have not yet been elucidated.

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0 h) and in after inoculation (6, 12 and 24 h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. Biological significance Here, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0 h) and in after inoculation (6, 12 and 24 h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Graphical abstract Improved and efficient metabolite extraction method for maximizing the number of metabolites identified from a single biological sample, compatible with DI-FTICR-MS-based metabolomics., Highlights • An improved metabolite extraction method for DI-FTICR metabolomics was developed. • The number of identified metabolites from grapevine leaves was maximized. • The extraction method allowed the extraction of polar and non-polar compounds. • Identified metabolites covered all major classes found in plants., In metabolomics there is an ever-growing need for faster and more comprehensive analysis methods to cope with the increase of biological studies. Direct infusion Fourier-transform ion cyclotron-resonance mass spectrometry (DI-FTICR-MS) is used in non-targeted metabolomics to obtain high-resolution snapshots of the metabolic state of a system. In any metabolic profiling study, the establishment of an effective metabolite extraction protocol is paramount. We developed an improved metabolite extraction method, compatible with DI-FTICR-MS-based metabolomics, using grapevine leaves. This extraction protocol allowed the extraction of polar and non-polar compounds, covering all major classes found in plants and increasing metabolome coverage.

Grapevine (Vitis vinifera L.), widely used for berry and wine production, is highly susceptible to the pathogenic oomycete Plasmopara viticola, the etiological agent of grapevine downy mildew disease. The method commonly used to prevent and control P. viticola infection relies on multiple applications of chemical fungicides. However, with European Union goals to lower the usage of such chemicals in viticulture there is a need to develop new and more sustainable strategies. The use of beneficial microorganisms with biocontrol capabilities, such as the arbuscular mycorrhizal fungi (AMF), has been pointed out as a viable alternative. With this study, we intended to investigate the effect of AMF colonization on the expression of P. viticola effectors during infection of grapevine. Grapevine plants were inoculated with the AMF Rhizophagus irregularis and, after mycorrhizae development, plants were infected with P. viticola. The expression of P. viticola RxLR effectors was analyzed by real-time PCR (qPCR) during the first hours of interaction. Results show that pre-mycorrhizal inoculation of grapevine alters the expression of several P. viticola effectors; namely, PvRxLR28, which presented decreased expression in mycorrhizal plants at the two time points post-infection tested. These results suggest that the pre-inoculation of grapevine with AMF could interfere with the pathogen’s ability to infect grapevine by modulation of pathogenicity effectors expression, supporting the hypothesis that AMF can be used to increase plant resistance to pathogens and promote more sustainable agriculture practices, particularly in viticulture.

Plants are sessile organisms being constantly under a wide array of environmental pressures. Their resistance against biotic stress is regulated by phytohormones, of which jasmonic acid (JA) plays an important role against necrotrophic pathogens and herbivorous insects whereas salicylic acid (SA) plays a crucial role in plant defence against biotrophic and hemi-biotrophic pathogens, as well as in the establishment of systemic acquired resistance. Plasmopara viticola is a biotrophic oomycete responsible for one of the most important diseases in viticulture. Recent studies have shown that JA-signalling may be playing an important role on grapevine resistance against this biotrophic pathogen. Expression of enzymes associated to JA biosynthesis (LOX2, AOS, AOC, OPR3), activation (JAR1) and signalling (COI1) was analysed in two Vitis vinifera genotypes with different degrees of resistance towards P. viticola. Our results provide the first clues for a JA-signalling role in grapevine defence against this fungal biotroph.

We investigated whether the performance of cork oak under drought could be improved by colonization with the ectomycorrhizal fungus Pisolithus tinctorius. Results show that inoculation alone had a positive effect on plant height, shoot biomass, shoot basal diameter, and root growth. Under drought, root growth of mycorrhizal plants was significantly increased showing that inoculation was effective in increasing tolerance to drought. In accordance, mycorrhizal plants subjected to drought showed less symptoms of stress when compared to non-mycorrhizal plants, such as lower concentration of soluble sugars and starch, increased ability to maintain fatty acid content and composition, and increased unsaturation level of membrane lipids. After testing some of the mechanisms suggested to contribute to the enhanced tolerance of mycorrhizal plants to drought, we could not find any by which Pisolithus tinctorius could benefit cork oak, at least under the drought conditions imposed in our experiment. Inoculation did not increase photosynthesis under drought, suggesting no effect in sustaining stomatal opening at low soil water content. Similarly, plant water status was not affected by inoculation suggesting that P. tinctorius does not contribute to an increased plant water uptake during drought. Inoculation did increase nitrogen concentration in plants but it was independent of the water status. Furthermore, no significant mycorrhizal effect on drought-induced ROS production or osmotic adjustment was detected, suggesting that these factors are not important for the improved drought tolerance triggered by P. tinctorius.

One of the most remarkable pollination strategy in orchids biology is pollination by sexual deception, in which the modified petal labellum lures pollinators by mimicking the chemical (e.g. sex pheromones), visual (e.g. colour and shape/size) and tactile (e.g. labellum trichomes) cues of the receptive female insect species. The present study aimed to characterize the transcriptional changes occurring after pollination in the labellum of a sexually deceptive orchid (Ophrys fusca Link) in order to identify genes involved on signals responsible for pollinator attraction, the major goal of floral tissues. Novel information on alterations in the orchid petal labellum gene expression occurring after pollination demonstrates a reduction in the expression of alkene biosynthetic genes using O. fusca Link as the species under study. Petal labellum transcriptional analysis revealed downregulation of transcripts involved in both pigment machinery and scent compounds, acting as visual and olfactory cues, respectively, important in sexual mimicry. Regulation of petal labellum senescence was revealed by transcripts related to macromolecules breakdown, protein synthesis and remobilization of nutrients.

In order to contribute to the knowledge on the genes involved in the early steps of ectomycorrhiza development, the transcriptional response of Castanea sativa (European chestnut) during the initial contact (6 and 12 h) with Pisolithus tinctorius was analysed by microarray. This study revealed that among the regulated plant genes, a substantial number of up-regulated transcripts showed homology with genes encoding for proteins involved in stress and defense responses, (a cystatin, a cystatin-like protein, a defensin and a universal stress protein). Early contact with the fungal mycelium also altered expression of genes that are putatively involved in cellular processes like signal transduction and communication (receptor kinase-related protein), protein fate (papain-like cystein proteinase), and water transport facilitation (water channel MipK protein). Expression profiling of the early contact between C. sativa and P. tinctorius revealed that changes in gene expression occur few hours after contact, long before the development of a functional mycorrhiza. The induction of genes involved in stress and defense suggests that the host plant reacts rapidly to the presence of the mycobiont eliciting a defense programme similar to that described for pathogenic interactions. Another plant response was the repression of genes normally implicated in water stress accounting for a water stress relief due to the initial contact with the ectomycorrhizal fungus.

This study aims at examining the expression of genes on the mammary gland, corresponding to various levels of adaptation or acclimatization to environmental stress. The authors utilized 18 cows from three genetic groups, Holstein Brazil (HB), Gyr (GG) and Gyrolando (GH ), all in the same stage of lactation, and subjected them to the same management conditions. Venous and arterial blood were collected to determine the hormonal profile and blood chemistry. Mammary gland tissue was used for transcriptomic studies. Prolactin and GH plasmatic concentrations were higher in Holstein animals. There were no differences in IGF-1 concentrations among the experimental groups. T 3 concentrations were similar among the Holstein and Gyr groups. From the 4608 transcripts in the BLO-Bovine EST (Michigan State University, US) databank that were used in this experiment, 105 differentially expressed genes were identified in at least one of the groups. Among these, the authors highlighted 14 genes that were related to the structure of the mammary gland (CRDGF, CD97, GH, endoglin, LTF, INPP, PTP), to response to thermal stress (Crh_11, v-Fos, Cdc37) and to milk protein (RPL35, κ-casein, β-casein, α-s2-casein). Eight of these were validated through real-time polymerase chain reaction. The HB animals, in comparison with the GH and Gyr groups, presented up-regulated genes associated with epithelium cellular differentiation and proliferation, milk productivity and decreased heat stress tolerance. Gyr animals presented up-regulated transcripts associated with cellular defence, apoptosis processes and increased tolerance to heat stress. The GH group showed intermediary results compared with the other two groups. Keywords: Dairy cow, mammary gland, transcriptomic, thermic stress

The European chestnut, an important forest species for the economy of Southern Europe, covers an area of 2.53 million hectares, including 75,000 ha devoted to fruit production. Castanea sativa is declining due to ink disease caused by Phytophthora cinnamomi. To elucidate chestnut defense mechanisms to ink disease, we compared the root transcriptome of the susceptible species C. sativa and the resistant species C. crenata after P. cinnamomi inoculation. Four cDNA libraries were constructed, two of them included root samples from C. sativa, inoculated and non-inoculated and the other two libraries comprised samples from C. crenata at identical conditions. Pyrosequencing produced 771,030 reads and assembly set up 15,683 contigs for C. sativa and 16,828 for C. crenata. GO annotation revealed terms related to stress as “response to stimulus”, “transcription factor activity” or “signaling” for both transcriptomes. Differential gene expression analysis revealed that C. crenata involved more genes related with biotic stress upon pathogen inoculation than C. sativa. Those genes for both species are involved in regulation of plant immune response and stress adaptation and recovery. Furthermore, it is suggested that both species recognize the pathogen attack; however, the resistant species may involve more genes in the defense response than the susceptible species. RNA-seq enabled the selection of candidate genes for ink disease resistance in Castanea. The present data is a valuable contribution to the available Castanea genomic resources and constitutes the basis for further studies.

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response., This project was funded by "Fundacao para a Ciencia e a Tecnologia" (FCT) within a National Consortium (COEC - Cork Oak ESTs Consortium) that supported 12 sub-projects (SOBREIRO/033, 035, 014, 034, 015, 017, 038, 019, 029, 039, 030, 036/2009). The authors further wish to acknowledge FCT for ten doctoral (BD) and post doctoral (BPD) fellowships (Tania Almeida: SFRH/BD/44410/2008, Tiago Capote: SFRH/BD/69785/2010, Ines Chaves: SFRH/BPD/20833/2004, Ana S. Fortunato: SFRH/BPD/47563/2008, Marilia Horta: SFRH/BPD/63213/2009, Liliana Marum: "SFRH/BPD/47679/2008, Andreia Miguel: SFRH/BD/44474/2008, Margarida Rocheta: SFRH/BPD/64905/2009, Tatiana E. Santo: SFRH/BD/47450/2008, Monica Sebastiana: SFRH/BPD/25661/2005). Andreas Bohn, Nelson J.M. Saibo, Rita Teixeira were supported by the Programa Ciencia 2007, financed by POPH (QREN) and Isabel A. Abreu, Susana Araujo, Dora Batista, A. Margarida Fortes, Jorge A.P. Paiva, Sonia Goncalves by Programa Ciencia 2008, also funded by POPH (QREN). A Margarida Santos was funded through iBET (PEst-OE/EQB/LA0004/2011). Maintenance of the CorkOakDB is supported by the Instituto Gulbenkian de Ciencia.

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-2-210) contains supplementary material, which is available to authorized users.

Grapevine species (Vitis sp.) are prone to several diseases, fungi being the major pathogens compromising its cultivation and economic profit around the world. Knowledge of the complexity of mechanisms responsible for resistance to fungus infection of cultivars, such as Regent, is necessary for strategies to be defined which will improve resistance in highly susceptible crop species. Transcript and metabolic profiles of the Vitis vinifera cultivars Regent and Trincadeira (resistant and susceptible to fungi, respectively) were analysed by cDNA microarray, quantitative real-time PCR, and nuclear magnetic resonance spectroscopy. The integration of datasets obtained through transcriptome and metabolome analysis revealed differences in transcripts and metabolites between both cultivars. These differences are probably associated with the innate resistance of Regent towards the mildews. Several transcripts related to stress and defence, namely a subtilisin-like protease, phenylalanine ammonia lyase, S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in Regent suggesting an intrinsic resistance capability of this cultivar. A metabolic profile revealed an accumulation of compounds such as inositol and caffeic acid, which are known to confer resistance to fungi. The differences in transcripts and metabolites detected are discussed in terms of the metabolic pathways and their possible role in plant defence against pathogen attack, as well as their potential interest to discriminate among resistant and susceptible grapevine cultivars.

Transcriptional changes in Pisolithus tinctorius leading to ectomycorrhizal formation in P. tinctorius– Castanea sativa were investigated using a 12-h fungal interaction in vitro system. Using a 3107-cDNA clone microarray, 34 unique expressed sequence tags (ESTs) were found to be differentially expressed. These ESTs represent 14 known genes, 5 upregulated and 9 downregulated, and 20 orphan sequences. Some transcripts of upregulated genes (with unknown function) were previously identified in other mycorrhizal Pisolithus spp. associations. ESTs for S-adenosyl-l-homocysteine hydrolase and several orphan sequences were identified in our system. The identified transcript of downregulated genes involved hydrophobins, 5S, 18S, and 28S ribosomal RNA genes, large subunits of ribosomal RNA (mitochondrial gene), and two types of heat shock proteins. This study demonstrates the high complexity of molecular events involved in the preinfection steps and suggests the utilization of different fungal gene repertories before ectomycorrhizal formation. These data constitute a first contribution for the molecular understanding of early signaling events between P. tinctorius and C. sativa roots during ectomycorrhizal formation.

Background Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. Results An integrated molecular and metabolomic approach was used to investigate global gene expression and metabolic responses during development of hop's organogenic nodules. Transcript profiling using a 3,324-cDNA clone array revealed differential regulation of 133 unigenes, classified into 11 functional categories. Several pathways seem to be determinant in organogenic nodule formation, namely defense and stress response, sugar and lipid metabolism, synthesis of secondary metabolites and hormone signaling. Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, lipid and sugar metabolism and secondary metabolism in organogenic nodule formation. Conclusion The expression profile of genes pivotal for energy metabolism, together with metabolites profile, suggested that these morphogenic structures gain energy through a heterotrophic, transport-dependent and sugar-degrading anaerobic metabolism. Polyamines and auxins are likely to be involved in the regulation of expression of many genes related to organogenic nodule formation. These results represent substantial progress toward a better understanding of this complex developmental program and reveal novel information regarding morphogenesis in plants.

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the "symbiosis toolkits" and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19,552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis., This work was funded by the Portuguese Foundation for Science and Technology (www.fct.pt) in the frame of the project Cork Oak EST Consortium SOBREIRO/0034/2009. Post-doc grant to MS was supported by the Portuguese Foundation for Science and Technology (SFRH/BPD/25661/2005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.