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Plant growth promoting rhizobacteria (PGPR) are in increasing demand due to their role in promoting sustainable practices, not only in agriculture but also in forestry. Keeping in mind the future application of PGPR for increasing cork oak sustainability, the aim of this study was to find cork oak PGPR isolates with increased nutrient solubilisation traits, able to promote root morphological changes and/or antagonize cork oak bark phytopathogens. Soils from three cork oak forests with distinct bioclimates (humid, semi-humid and semi-arid) were used for isolating bacteria. From the 7634 colony-forming units, 323 bacterial isolates were biochemically assayed for PGPR traits (siderophores production, phosphate solubilizing and organic acids production), and 51 were found to display all these traits. These PGPR were able to induce root morphological changes on Arabidopsis thaliana, like suppression of primary root growth, increase of lateral roots or root hairs formation. However, the most proficient PGPR displayed specific ability in changing a single root morphological trait. This ability was related not only to bacterial genotype, but also with the environment where bacteria thrived and isolation temperature. Bacteria from semi-arid environments (mainly Bacillus megaterium isolates) could hold a promising tool to enhance plant development. Other isolates (Serratia quinivorens or B. cereus) could be further explored for biocontrol purposes., This work was supported by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundação para a Ciência e a Tecnologia) in the framework of the projects SuberControl (PTDC/ASP-SIL/28635/2017), BioISI (UIDB/04046/2020) and CIMO (UIDB/00690/2020).

Ectomycorrhizal fungi are crucial for forests sustainability. For Castanea sativa, ectomycorrhizal fungus Pisolithus tinctorius is an important mutualist partner. Saprotrophic fungi Hypholoma fasciculare, although used for biocontrol of Armillaria root disease, it negatively affected the interaction between the P. tinctorius and plant host roots, by compromise the formation of P. tinctorius-C. sativa mycorrhizae. In this work, fungal morphology during inhibition of H. fasciculare against P. tinctorius was elucidated. P. tinctorius growth was strongly affected by H. fasciculare, which was significantly reduced after six days of co-culture and become even more significant through time. During this period, P. tinctorius developed vesicles and calcium oxalate crystals, which were described as mechanisms to stress adaption by fungi. H. fasciculare produced different volatile organic compounds in co-cultures over time and differ between single or in dual-species. H. fasciculare highly produced sesquiterpenes (namely, alpha-muurolene) and nitrogen-containing compounds, which are recognised as having antimicrobial activity., This work was funded by FEDER through the Operational Competitiveness Program (COMPETE) and by Portuguese national funds through the Foundation for Science and Technology (FCT) within the scope of project PTDC/AGR-AAM/099556/2008, UID/MULTI/04046/2013, and the CIMO (UIDB/00690/2020) and the Applied Molecular Biosciences Unit-UCIBIO (UID/Multi/04378/2020). Nathalie Moreira acknowledge FCT for her grant, SFRH/BDP/109668/2015.

Plant-inhabiting microorganisms interact directly with each other affecting disease progression. However, the role of host plant and plant habitat in shaping pathobiome composition and their implication for host susceptibility/resistance to a particular disease are currently unknown. For the elucidation of these questions, both epiphytic and endophytic bacterial communities, present in asymptomatic and symptomatic twigs from olive cultivars displaying different susceptibilities to olive knot (OK) disease, were investigated using culturing methods. OK disease was the main driver of the bacterial community, causing changes on their diversity, abundance and composition. OK disease effect was most notorious on OK-susceptible cultivar and when considering the endophytic communities. Plant habitat (epiphytes vs. endophytes) also contributed to the bacterial community assembling, in particular on symptomatic twigs (knots) of OK-susceptible cultivar. In contrast, host cultivar had little effect on the bacterial community composition, but OK-symptomatic twigs (knots) revealed to be more affected by this driver. Overall, the pathobiome seems to result from an intricate interaction between the pathogen, the resident bacteria, and the plant host. Specific bacterial genera were associated to the presence or absence of OK disease in each cultivar. Their ability to trigger and/or suppress disease should be studied in the future., The authors are grateful to the FEDER and Foundation for Science and Technology (FCT, Portugal) under Programme PT2020 for financial support to CIMO (UIDB/00690/2020) and BioISI (UID/MULTI/04046/2013), as well as the Horizon 2020, the European Union's Framework Programme for Research and Innovation, for financial support the project PRIMA/0002/2018 INTOMED -Innovative tools to combat crop pests in the Mediterranean. D. Mina thanks FCT, POPH-QREN and FSE for SFRH-BD-105341/2014 grant.

The common bean (Phaseolus vulgaris L.) is the most consumed legume and the major protein source for poor populations, especially in developing Afro-Asian countries. Common bean has a high nutritional value, being rich in protein, carbohydrates, essential fatty acids, vitamins, minerals, and fibers. Besides these nutrients, common bean is also enriched in bioactive compounds, such as polyphenols, mainly flavonoids. These compounds reveal important antioxidant activities through their capacity to interfere with oxidative processes and consequently exhibit antiinflammatory activity. Common bean also reveals anticarcinogenic and antimutagenic activities. Accordingly the consumption of common bean has been associated with the prevention and control of several age-related problems, such as diabetes, obesity, cardiovascular and gastrointestinal diseases, and cancer. This chapter focuses on the bioactivities present in common bean grains that make this legume an excellent functional food. Therefore the increase in common bean consumption, in traditional or nutraceutical forms, should be encouraged for improving human health.

Cowpea is a grain legume native from Africa and is a primary source of protein for millions of people in sub-Saharan Africa and other parts of the developing world. The main important characteristics of this crop include a good protein quality with a high nutritional value, its nitrogen-fixing ability, and an ability to be more drought- and heat-tolerant than most of its legume relatives. In a research perspective, studies of cowpea are relatively scarce, despite its relevance to agriculture in the developing world and its resilience to stress. The present review provides an overview of different aspects of cowpea, with a special emphasis on the molecular markers for assessing genetic diversity, as well as on biochemical and transcriptomic data with respect to evaluating cowpea drought stress tolerance. The integration of both datasets will be useful for the improvement of cowpea because research on drought stress tolerance is of major interest for this crop in a challenging environment. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.scienta.2018.11.082., Global warming has an increasing impact on the availability of water for agriculture. Crops tolerant to high temperatures and drought, such as cowpea (Vigna unguiculata L. Walp.), have an added value in the near future. The main objective of this study was to evaluate the effect of drought on seed germination and seedling emergence of cowpea genotypes, in order to screen the most tolerant genotypes. Seeds from 58 cowpea genotypes all over the world were submitted to two stress conditions, induced by PEG-6000 (corresponding to osmotic potentials of -0.75 bars and -1.5 bars). Germination and seedling growth parameters, vigor index and proline content were determined to assess drought tolerance. The results revealed significant differences of all parameters among genotypes after treatments and interaction of both. Water stress caused a general decrease in germination and seedling growth, while an increase in proline content was observed. A high variation of drought responses were detected among genotypes, being possible to select seven genotypes (C11, C18, C44, C46, C47, C50 and C54) as tolerant to drought at germination stage. These results will be useful to select the best suitable parents for insertion in future breeding programs., This study was supported by EUROLEGUME project. This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613781. European Investment Funds by FEDER/COMPETE/POCI - Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the projects UID/AGR/04033/2013 and UID/AGR/04046/2013.

Plants are able to interact with plentiful bacteria resulting in a number of positive or negative outcomes for plant health. The ecological balance between pathogens and beneficial bacteria could be strategically disturbed and manipulated for improving host plant protection. As bacterial communities present in the phyllosphere of herbaceous plants have been largely studied, a number of biocontrol agents for controlling host diseases are already identified and used with promising results. A few studies on the use of phyllosphere biocontrol agents on woody crop tree plants have revealed encouraging results toward a future where plant disease control could be attained without the application of chemical compounds. In addition to the use of biocontrol agents, disease suppression can be achieved by the manipulation of microbial communities through plant management practices. In this review, an overview of the available knowledge on phyllosphere bacterial communities of woody tree crop species is provided, giving special emphasis to the structural differences of bacterial communities living on and within important tree crop species. Studies and challenges on the application and/or manipulation of these bacteria under in planta conditions are discussed, disclosing new sustainable ways for dealing with woody crop diseases.

In nature, pathogens live and interact with other microorganisms on plant tissues. Yet, the research area exploring interactions between bacteria-fungi and microbiota-plants, within the context of a pathobiome, is still scarce. In this study, the impact of olive knot (OK) disease caused by the bacteria Pseudomonas savastanoi pv. savastanoi (Psv) on the epiphytic and endophytic fungal communities of olive tree twigs from three different cultivars, was investigated in field conditions. The ITS-DNA sequencing of cultivable fungi, showed that OK disease disturbs the resident fungal communities, which may reflect changes in the habitat caused by Psv. In particular, a reduction on epiphyte abundance and diversity, and changes on their composition were observed. Compared to epiphytes, endophytes were less sensitive to OK, but their abundance, in particular of potential pathogens, was increased in plants with OK disease. Host genotype, at cultivar level, contributed to plant fungal assembly particularly upon disease establishment. Therefore, besides fungi - Psv interactions, the combination of cultivar - Psv also appeared to be critical for the composition of fungal communities in olive knots. Specific fungal OTUs were associated to the presence and absence of disease, and their role in the promotion or suppression of OK disease should be studied in the future., This research was partially supported by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds through FCT (Fundacao para a Ciencia e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012. This work was supported by FCT under the project UID/MULTI/04046/2013. T. Gomes thanks FCT, POPH-QREN and FSE for PhD SFRH/BD/98127/2013 grant; and also the COST Action FA1405 for a short-term scientific mission (STSM) grant.

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.micres.2020.126693., A wide array of fungal endophytes is known to inhabit plant tissues and were recently recognized as essential for plant health. A better description of the scarcely known endophyte microbiota in olive tree phyllosphere is the first step for elucidating the microbial interactions that lead to olive disease establishment. In this work, the fungal endophytic community of the phyllosphere of different olive tree cultivars (Cobrancosa, Galega vulgar, Madural, Picual, Verdeal Transmontana) is revealed by using a metabarcoding strategy targeting ITS1 barcode. A total of 460 OTUs were obtained, increasing the broad view of fungal endophytes inhabiting the olive tree phyllosphere, in particular yeast endophytes. New endophytes were persistently found in all cultivar tissues. Different olive tree cultivars depicted distinct endophyte communities. Olive cultivars exhibited dissimilar amounts of fungi with distinct ecological functions, which could explain at least in part their differential susceptibility/tolerance to olive diseases., This work was supported by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundacao para a Ciencia e a Tecnologia) in the framework of the projects EXCL/AGR-PRO/0591/2012, PTDC/AGR-PRO/4354/2012, and PTDC/ASP-PLA/31133/2017, as well as BioISI (UIDB/04046/2020) and Mountain Research Center - CIMO (UIDB/00690/2020).

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.02051/full#supplementary-material, Olive knot (OK) is a widespread bacterial disease, caused byPseudomonas savastanoipv.savastanoi(Pss), which currently has not effective control methods. The use of naturally occurring microbial antagonists, such as bacteria, as biocontrol agents could be a strategy to manage this disease. The objective of this work was to select bacteria from olive tree phyllosphere able to antagonizePssusingin vitroandin plantaexperiments. The elucidation of their modes of action and the potential relationship between antagonism and bacteria origin has been investigated, as well. To this end, 60 bacterial isolates obtained from the surface and inner tissues of different organs (leaves, twigs, and knots), from two olive cultivars of varying susceptibilities to OK, were screened for theirin vitroantagonistic effect againstPss. A total of 27 bacterial strains were able to significantly inhibitPssgrowth, being this effect linked to bacteria origin. Strains from OK-susceptible cultivar and colonizing the surface of plant tissues showed the strongest antagonistic potential. The antagonistic activity was potentially due to the production of volatile compounds, siderophores and lytic enzymes.Bacillus amyloliquefaciensP41 was the most effective antagonistic strain and their capacity to control OK disease was subsequently assayed usingin plantaexperiments. This strain significantly reduces OK disease severity (43.7%), knots weight (55.4%) and population size ofPss(26.8%), while increasing the shoot dry weight (55.0%) and root water content (39.6%) ofPss-infected olive plantlets. Bacterial isolates characterized in this study, in particularB. amyloliquefaciensP41, may be considered as promising biocontrol candidates for controlling OK disease., This work was funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade), national funds through FCT (Fundacao para a Ciencia e a Tecnologia) and by Horizon 2020, the European Union's Framework Programme for Research and Innovation, within the project PRIMA/0002/2018 (INTOMED - Innovative tools to combat crop pests in the Mediterranean), and the Mountain Research Center - CIMO (UIDB/00690/2020 and UIDB/04046/2020).

Variation on bacterial communities living in the phyllosphere as epiphytes and endophytes has been attributed to plant host effects. However, there is contradictory or inconclusive evidence regarding the effect of plant genetics (below the species' level) and of plant tissue type on phyllosphere bacterial community assembly, in particular when epiphytes and endophytes are considered simultaneously. Here, both surface and internal bacterial communities of two olive (Olea europaea) cultivars were evaluated in twigs and leaves by molecular identification of cultivable isolates, with an attempt to answer these questions. Overall,Proteobacteria,ActinobacteriaandFirmicuteswere the dominant phyla, being epiphytes more diverse and abundant than endophytes. Host genotype (at cultivar level) had a structuring effect on the composition of bacterial communities and, in a similar way, for both epiphytes and endophytes. Plant organ (leaf vs. twig) control of the bacterial communities was less evident when compared with plant genotype and with a greater influence on epiphytic than on endophytic community structure. Each olive genotype/plant organ was apparently selective towards specific bacterial operational taxonomic units (OTUs), which may lead to specific feedbacks on fitness of plant genotypes. Bacterial recruitment was observed to happen mainly within epiphytes than in endophytes and in leaves as compared with twigs. Such host specificity suggested that the benefits derived from the plant-bacteria interaction should be considered at genetic levels below the species., The authors are grateful to the FEDER and Foundation for Science and Technology (FCT, Portugal) under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019) and BioISI (UID/MULTI/04046/2013), as well as the Horizon 2020, the European Union’s Framework Programme for Research and Innovation, for financial support the project PRIMA/0002/2018 INTOMED—Innovative tools to combat crop pests in the Mediterranean. D. Mina thanks FCT, POPH-QREN and FSE for SFRH-BD-105341/2014 grant.

The dynamics of Xylella fastidiosa infections in the context of the endophytic microbiome was studied in field-grown plants of the susceptible and resistant olive cultivars Kalamata and FS17. Whole metagenome shotgun sequencing (WMSS) coupled with 16S/ITS rRNA gene sequencing was carried out on the same trees at two different stages of the infections: In Spring 2017 when plants were almost symptomless and in Autumn 2018 when the trees of the susceptible cultivar clearly showed desiccations. The progression of the infections detected in both cultivars clearly unraveled that Xylella tends to occupy the whole ecological niche and suppresses the diversity of the endophytic microbiome. However, this trend was mitigated in the resistant cultivar FS17, harboring lower population sizes and therefore lower Xylella average abundance ratio over total bacteria, and a higher α-diversity. Host cultivar had a negligible effect on the community composition and no clear associations of a single taxon or microbial consortia with the resistance cultivar were found with both sequencing approaches, suggesting that the mechanisms of resistance likely reside on factors that are independent of the microbiome structure. Overall, Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes dominated the bacterial microbiome while Ascomycota and Basidiomycota those of Fungi., This research was funded by the EU H2020 Research Project XF-ACTORS "Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy" (Grant Agreement 727987) and by the Mountain Research Center-CIMO (UIDB/00690/2020) and BioISI (UIDB/04046/2020). The Funding Agencies are not responsible for any use that may be made of the information it contains.

Climate changes comprise increasing global temperature and water cycle deregulation (precipitation storms and long dry seasons). Many affected ecosystems are located within the Mediterranean basin, where cork oak (Quercus suber L.) is one of the most important forest ecosystems. Despite cork oak tolerance to drought, the decrease of water availability and increase of temperature is causing a serious decline of cork oak populations. In the present work, the bacterial community of cork oak soils was assessed by metabarcoding using Illumina Miseq. Soils from seven independent cork oak forests were collected along a climate gradient. In all forest soils, Proteobacteria and Actinobacteria were the richest and more abundant bacteria. Acidobacteria also presented a high relative abundance, and Chloroflexi was a rich phylum. The soil bacterial community diversity and composition was strongly affected by the climatic region where cork oak resides and specific bacterial taxa were differently affected by precipitation and temperature. Accordingly, cork oak bacterial communities clustered into three distinct groups, related with humid, sub-humid and arid/semi-arid climates. Driest and warmer forests presented more diverse bacterial communities than humid and coolest forests. However, driest climates presented more homogenous bacterial communities among forests than humid climates. Climate (mainly precipitation) revealed to be the strongest driver leading to significant variations of bacterial community profiles. The most impacted bacterial taxa by climatic variables were Proteobacteria, in particular Gammaproteobacteria and Deltaproteobacteria, Chloroflexi, and Firmicutes. Humid forests presented mainly Acidobacteria as good indicators of climate, whereas Actinobacteria members were better indicators for arid forests (mainly Gaiellales and Frankiales). Some indicator species for different climate conditions were members of the bacterial core of cork oak stands (7% of the total bacterial community). Taken together, different, This work was supported by FEDER through the Operational Competitiveness Program (COMPETE) and by Portuguese national funds through the Foundation for Science and Technology (FCT) within the scope of the project POCI-01-0145-FEDER-028635; FCT/MCTES/PIDDAC (Portugal) under the project (PEst-OE/BIA/UI4046/2014; UID/MULTI/04046/2013) and PhD grant to F.R. (SFRH/BD/86519/2012).

The online version of this article (https://doi.org/10.1007/s00248-018-1161-9) contains supplementary material, which is available to authorized users., The diversity and factors influencing fimgal assemblages in phyllosphere of Mediterranean tree species have been barely studied, especially when endophytic and epiphytic communities are simultaneously considered. In this work, the endophytic and epiphytic fungal communities from olive tree phyllosphere were studied. This tree species is natural from the Mediterranean region and adapted to grow under adverse climatic conditions. The main objectives were to determine whether there are differences between both fungal communities and to examine whether different abiotic (climate-related) and biotic (plant organs) factors play a pivotal role in structuring these communities. Both communities differed in size and composition, with epiphytic community being richer and more abundant, displaying also a dominance of melanized fungi. Season was the major driver of community composition, especially of epiphytes. Other drivers shaping epiphytes were wind speed and temperature, while plant organ, rainfall, and temperature were the major drivers for endophytic composition. In contrast, canopy orientation caused slight variations in community composition of fungi, but with distinct effects in spring and autumn seasons. In conclusion, epiphytic and endophytic communities are not driven by the same factors. Several sources of variation undergo complex interactions to form and maintain phyllosphere fungal community in Mediterranean climates. Climatic parameters have influence on these fungal communities, suggesting that they are likely to be affected by climate changes in a near future., This work is funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundacao para a Ciencia e a Tecnologia) within the framework of the project EXCL/AGR-PRO/0591/2012. T. Gomes thanks FCT, POPH-QREN, and FSE for PhD SFRH/BD/98127/2013 grant.

Cowpea (Vigna unguiculata L. Walp) is an important legume crop due to its high protein content, adaptation to heat and drought and capacity to fix nitrogen. Europe has a deficit of cowpea production. Knowledge of genetic diversity among cowpea landraces is important for the preservation of local varieties and is the basis to obtain improved varieties. The aims of this study were to explore diversity and the genetic structure of a set of Iberian Peninsula cowpea accessions in comparison to a worldwide collection and to infer possible dispersion routes of cultivated cowpea., This study was supported by EUROLEGUME project. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613781. European Investment Funds by FEDER/COMPETE/ POCI – Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT – Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013. MMA was partially supported by the Feed the Future Innovation Lab for Climate Resilient Cowpea (USAID Cooperative Agreement AID-OAA-A-13-00070), which is directed by TJC. The funding entities had no role in the design of the study, collection, analysis and interpretation of data, or in writing the manuscript., info:eu-repo/semantics/publishedVersion

Drought impact on plants is an increasing concern under the climate change scenario. Cowpea (Vigna unguiculata L. Walp.) is considered as one of the most tolerant legume crops to drought, being the search for the best well-adapted genotypes crucial to face the future challenges. Different approaches have been used for differentiating plant responses to drought stress. Plants of four cowpea genotypes were submitted to three watering regimens (a severe and moderate drought stress, and well-watered control) during 15 days, and several physiological, biochemical and molecular parameters were evaluated. Stressed plants revealed commonly-described drought stress characteristics, but not all assayed parameters were useful for discriminating plants with different drought severities or genotypes. The analyses which have contributed most to genotype discrimination were those related with stomatal function, and biochemical markers such as proline and anthocyanin contents. Antioxidant enzymes activities and related genes expression did not differed among genotypes or upon drought stress treatments, suggesting that scavenging enzymes are not involved in the differential ability of cowpea plants to survive under drought stress. This information will be useful to evaluate and use genetic resources, as well as design strategies for breeding cowpea resistance to drought stress., This study was supported by EUROLEGUME project. This project has received funding from: the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613781; National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019; and the project PEstOE/BIA/UI4046/2014; UID/MULTI/04046/2013.

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time., Authors are grateful to Fundacao para a Ciencia e Tecnologia (FCT) for financial support (Project PTDC/AGR-AAM/099556/2008 and Pest-C/BIA/UI4050/2011, FEDER-COMPETE, FCT, Portugal)., info:eu-repo/semantics/publishedVersion

Quercus suber (cork oak) is a West Mediterranean species of key economic interest, being extensively explored for its ability to generate cork. Like other Mediterranean plants, Q. suber is significantly threatened by climatic changes, imposing the need to quickly understand its physiological and molecular adaptability to drought stress imposition. In the present report, we uncovered the differential transcriptome of Q. suber roots exposed to long-term drought, using an RNA-Seq approach. 454-sequencing reads were used to de novo assemble a reference transcriptome, and mapping of reads allowed the identification of 546 differentially expressed unigenes. These were enriched in both effector genes (e.g., LEA, chaperones, transporters) as well as regulatory genes, including transcription factors (TFs) belonging to various different classes, and genes associated with protein turnover. To further extend functional characterization, we identified the orthologs of differentially expressed unigenes in the model species Arabidopsis thaliana, which then allowed us to perform in silico functional inference, including gene network analysis for protein function, protein subcellular localization and gene co-expression, and in silico enrichment analysis for TFs and cis-elements. Results indicated the existence of extensive transcriptional regulatory events, including activation of ABA-responsive genes and ABF-dependent signaling. We were then able to establish that a core ABA-signaling pathway involving PP2C-SnRK2-ABF components was induced in stressed Q. suber roots, identifying a key mechanism in this species' response to drought., This work was supported by FEDER through the Operational Competitiveness Program – COMPETE – and by national funds through the Foundation for Science and Technology – FCT – within the scope of projects SOBREIRO/0033/2009 “Cork oak ESTs Consortium – Abiotic stress: drought, salt and oxidative stresses” and PTDC/AGRGPL/118505/2010“An integrated approach to identify stress-related regulatory genes in cork oak (SuberStress).” PHC was supported by FEDER through COMPETE, and by national funds (FCT) within the scope of project “SUMOdulator” [Refs. FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012]. HA was supported by the “Genomics and Evolutionary Biology” project, co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

A comprehensive understanding of the physiological responses of plants to extreme temperatures is essential for future strategies for plant improvement. Obvious advantages can result from the study of highly adapted plant species, such as the model tree Populus euphratica Olivier that naturally thrives under extreme temperatures, saline soils, and drought. The present paper addresses the issue of P. euphratica thermotolerance using a cell suspension model system. P. euphratica suspended cells were subjected to a range of temperatures (from 5 up to 75°C) for 20 min, and cultures were evaluated for cell viability and biomass content at specific time points. The results have shown that cell viability was only affected after a temperature stress higher than 40°C, although in these conditions it was observed that a cell growth increases after the recovery period. In contrast, a total decline in cell viability was observed in suspended cells treated at 50°C or higher temperatures, which did not show growth recovery capacity. Therefore, the known natural tolerance of P. euphratica to thermal stress was not observable at the cellular level. The greater susceptibility to high temperatures in suspended cells as compared to field plants suggests that high thermotolerance can only be achieved when cells are integrated into a tissue.

In Northeast of Portugal the macrofungal community associated to chestnut tree (Castanea sativa Mill.) is rich and diversified. Among fungal species, the ectomycorrhizal Pisolithus tinctorius and the saprotroph Hypholoma fasciculare are common in this habitat. The aim of the present work was to assess the effect of the interaction between both fungi on growth, nutritional status and physiology of C. sativa seedlings. In pot experiments, C. sativa seedlings were inoculated with P. tinctorius and H. fasciculare individually or in combination. Inoculation with P. tinctorius stimulated the plant growth and resulted in increased foliar-N, -P, and photosynthetic pigment contents. These effects were suppressed when H. fasciculare was simultaneously applied with P. tinctorius. This result could be related to the inhibition of ectomycorrhizal fungus root colonization as a result of antagonism or to the competition for nutrient sources. If chestnut seedlings have been previously inoculated with P. tinctorius, the subsequent inoculation of H. fasciculare 30 days later did not affect root colonization and mycorrhization benefits were observed. This work confirms an antagonistic interaction between ectomycorrhizal and saprotrophic fungi with consequences on the ectomycorrhizal host physiology. Although P. tinctorius is effective in promoting growth of host trees by establishing mycorrhizae, in the presence of other fungi it may not always be able to interact with host roots due to an inability to compete with certain fungi., Authors are grateful to Fundação para a Ciência e Tecnologia (FCT) for financial support (Project PTDC/AGR-AAM/099556/2008).

Up to now the higher capacity of proteoid roots to absorb inorganic phosphate from P i -poor soils has been related mainly to their increased root surface area and higher exudation of organic acids and phosphatases, while much less attention has been directed to their mechanisms of P i uptake. Here we report a characterization of the P i uptake kinetics of the proteoid root-forming species Hakea sericea Schrad. This Proteaceae is an Australian native, which is disseminating very fast through forests of the European south. Dense mats of proteoid roots were observed in the upper soil layers of the invaded area in Portugal where availability of P and N was shown to be very low. Plants grown hydroponically under low-P i also developed proteoid roots, and the proteoid clusters presented a major role in P i absorption in comparison to the non-proteoid portions of the root system as revealed by their higher 32 P i labeling. The 32 P i uptake by proteoid roots was dependent on H + gradient and yielded a biphasic kinetics, suggesting the involvement of H + /P i co-transport systems with K m values of 0.225 and 40.8 μM P i . The analogs phosphite (Phi) and arsenate, but not vanadate, inhibited competitively the P i absorption. Such biphasic P i uptake pattern with the highest affinity at submicromolar range is likely to be of critical importance for the capacity of this plant species to invade and proliferate throughout vast areas of nutrient-deprived soils.

Plants encode catalase (CAT; EC 1.11.1.6) as multigene families, which may reflect the multiple and diverse roles played by this enzyme. Catalases from higher plants can be subdivided into three distinct types, according to their phylogenetic relationship. However, there is not a specific correlation of phylogeny and function within these groups, as catalases from the same type can play different specific roles. We report on the characterization of two monocot catalase genes from Zantedeschia aethiopica (a C_3-type Araceae) and its expression analysis during spathe senescence and regreening. Z. aethiopica CAT2 appears to play an exclusive role in scavenging photorespiratory H_2O_2, but is more phylogenetically related to the unspecific Zea mays CAT3 and Oryza sativa CATA than to the photorespiratory-related Zea mays CAT2 and Oryza sativa CATC. Z. aethiopica CAT1 is more phylogenetically related to Zea mays CAT1 and Oryza sativa CATB and appears to have a dual role in scavenging glyoxysomal and peroxisomal H_2O_2., Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI/BD/16280/98 , SFRH/BD/12081/2003.

Since the sequencing of the Arabidopsis thaliana genome in 2000, plant researchers have faced the complex challenge of assigning function to thousands of genes. Functional discovery by in silico prediction or homology search resolved a significant number of genes, but only a minor part has been experimentally validated. Arabidopsis entry into the post-genomic era signified a massive increase in high-throughput approaches to functional discovery, which have since become available through publicly-available web-based resources. The present work focuses on an easy and straightforward strategy that couples data-mining to reverse genetics principles, to allow for the identification of new abiotic stress determinant genes. The strategy explores systematic microarray-based transcriptomics experiments, involving Arabidopsis abiotic stress responses. An overview of the most significant resources and databases for functional discovery in Arabidopsis is presented. The successful application of the outlined strategy is illustrated by the identification of a new abiotic stress determinant gene, HRR, which displays a heat stress-related phenotype after a loss-of-function reverse genetics approach., No competing financial interests exist. The present work was supported by Foundation for Science and Technology (POCTI/AGR/45462/2002). H. Azevedo (SFRH/BPD/17198/2004), J. Correia (SFRH/BD/16663/2004), J. Oliveira (SFRH/BD/38379/2007), S. Laranjeira (SFRH/BD/29778/2006), C. Barbeta (SFRH/BD/12081/2003) and V. Amorim-Silva (SFRH/BD/29778/2006) were supported by Foundation for Science and Technology.

For the ectomycorrhizal establishment, different signals must be exchanged between plants and fungal partners. After a brief description of the ectomycorrhiza formation, the present chapter highlights the recent works on the nature of the signal molecules involved in this type of plant–microbe interaction. Signals involved in the fungal spore germination and mycelium growth, including the chemoattraction of the mycelium by the root, hyphae–root adhesion, and penetration of the fungus into the root will be addressed. The signals associated to the development of ectomycorrhizal structures will also be overviewed. For the production of signals, formation of symbiotic structures (mantle and Hartig-net), and ectomycorrhizal functioning, a new pattern of gene expression and protein synthesis is required. Studies reporting the involvement of plant defense responses during the early stages of ectomycorrhiza development and the role of reactive oxygen species as signaling molecules will be reviewed.

Models of non-host resistance have failed to account for the pathogenicity of necrotrophic agents. During the interaction of Pinus pinaster (maritime pine) with the non-host necrotrophic pathogen Botrytis cinerea, the generation and scavenging of reactive oxygen species (ROS) and the induction of the hypersensitive response (HR) were analyzed. Elicitation of maritime pine suspended cells with B. cinerea spores resulted in the biphasic induction of ROS. The phase I oxidative burst was dependent on calcium influx, while the phase II oxidative burst also depended on NADPH oxidase, protein kinase activity, and de novo transcription and protein synthesis. A decline was observed in catalase (CAT) and superoxide dismutase (SOD) activity, together with the down-regulation of Fe-Sod1, chlCu, Zn-Sod1 and csApx1, suggesting a coordinated response towards a decrease in the ROS-scavenging capacity of maritime pine cells during challenge. Following the second oxidative burst, programmed cell death events characteristic of the HR were observed. The results suggest the ROS-mediated and cell-breach-independent activation of Type II non-host resistance during the P. pinaster-B. cinerea interaction.

Conventional RNA isolation methods optimised for pine seedlings have been shown to produce poor quality RNA when applied to needles of adult pine trees. We describe here a modified procedure to isolate high pure RNA from needles of thirty-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides and RNases. Major changes were the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by denaturing gel electrophoresis and by spectrophotometry (A260/A230 and A260/A280). Total RNA could be successfully used for poly(A)+-RNA isolation and cDNA library construction., Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/3194/2000.

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.

Senescence is the terminal phase in the life of a plant or a plant organ that leads ultimately to death. From many physiological, biochemical, and molecular studies of leaf senescence, it becomes clear that leaf cells undergo highly coordinated changes in cell structure, metabolism, and gene expression during senescence [1].

The spathe of Zantedeschia aethiopica (L.) Spreng. (Araceae) undergoes a senescence process soon after its formation, leading to the death if fructification doesn’t occur [1]. During fructification, spathe regreening occurs being a consequence of a senescence-inhibition process. It was suggested that spathe senescence inhibition and regreening at time of fructification is due to the action of cytokinins produced by fruits and translocated to the spathe [2–4].

In Plant Kingdom, one of the strategies for plants to survive is the spontaneous production of direct somatic embryos [1]. Examples can be found in angiosperms, both in dicots and monocots. In artificial conditions, somatic embryogenesis is achieved using different types of initial expiants and following different types of stress [for a review see, 1,8,9,11,12]. Although both direct and indirect somatic embryogenesis have been successfully achieved for many species [9], the knowledge on the early events occurring during the induction of embryogenesis is still scarce. Here, we describe some of the molecular and elemental changes occurring during the early stages of direct somatic embryogenesis using, as model plant, the woody species Camellia japonica. Our previous results shown that direct embryogenesis from camellia leaves is region specific [5]. This specificity was suggested to be related to pre-established elemental gradients, namely, of calcium, sodium and sulphur [4,6]. After the induction treatment, two types of response were detected: a “fast” and a slower one [6]. The first one, common to all parenchyma cells, was suggested to be a stress response, due to leaf excision and to the induction treatment. The following morphogenic or non-morphogenic responses, that were cell or explant-region specific, were suggested to be dependent on the cell-state of each cell at the time of leaf isolation [6]. Some data on the possible effect of stress on embryogenic induction is presented. The role of Ca2+, Mg2+ and competing polyamines (free and conjugated) on cell division and morphogenesis is also discussed.