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Quercus spp. have formed broad-leaved evergreen forests in the Hindu Kush and Himalayan regions of Pakistan. Seven species of the genus Quercus (Q. baloot Griff., Q. dilatata Royle., Q. glauca Thunb., Q. incana Roxb., Q. robur Linn., Q. semecarpifolia Smith., and Q. leucotrichophora A. Camus.) have been identified. These species have received little attention compared with other economically valuable plant species in Pakistan, which has been mainly linked to traditional medicine and the identification of phytonutrients to evaluate their bioactivities and toxicological effects. Quercus spp. are promising for commercial applications, so government policy should encourage their management and conservation. However, they are currently threatened by severe human activities and climate change. The goal of this review is to highlight the relevance of these forgotten species, describing overall aspects related to their distribution, morphology, traditional uses, phytochemical constituents, and threats. To date, no proper and comprehensive molecular studies on the populations of these species found in Pakistan have been conducted, which is a critical gap as molecular studies are essential for conservation and management strategies. Finally, we discuss future directions in molecular approaches for Quercus that follow the strategies that are being used for other species of the genus Quercus that are not found in Pakistan.

The holm oak (Quercus ilexL.) is the most representative species of the Mediterranean Basin and the agrosilvopastoral Spanish “dehesa” ecosystem. Being part of our life, culture, and subsistence since ancient times, it has great environmental and economic importance. More recently, there has been a renewed interest in using theQ. ilexacorn as a functional food due to its nutritional and nutraceutical properties. However, the holm oak and its related ecosystems are threatened by different factors, with oak decline syndrome and climate change being the most worrying on the short and medium term. Breeding programs informed by selection of elite genotypes seems to be the only plausible biotechnological solution to rescue populations under threat. To achieve this and other downstream analyses, we need a high-qualityQ. ilexreference genome. Here, we introduce the first draft genome assembly ofQ. ilexusing long-read sequencing (PacBio). The assembled nuclear haploid genome has 530 contigs totaling 842.2 Mbp (N50 = 3.3 Mbp), of which 448.7 Mb (53%) are repetitive sequences. We annotated 39,443 protein-coding genes and Benchmarking Universal Single-Copy Orthologs analysis detected 412 out of 425 expected complete and single-copy genes (94.80%) within theQ. ilexgenome. The chloroplast genome size was 142.3 Kbp with 149 protein-coding genes successfully annotated. This first draft should allow for the validation of - omics data as well as the identification and functional annotation of genes related to phenotypes of interest such as those associated to resilience against oak decline syndrome and climate change, higher acorn productivity and nutraceutical value.

The holm oak (

(1) Background: Cadmium is a toxic heavy metal that is widely distributed in water, soil, and air. It is present in agrochemicals, wastewater, battery waste, and volcanic eruptions. Thus, it can be absorbed by plants and enter the trophic chain.

Background Pollen food allergy syndrome (PFAS) related to PR10 from vegetables is common in northern Europe, whereas in Mediterranean countries PFAS has been preferentially associated with profilins. However, there are pollen-allergic patients reactive to Bet v 1 in birch-free regions. Since it cannot be the primary sensitizer, there has to be another culprit. Quercus ilex is a good candidate as it belongs to the order Fagales. This order includes trees with highly sensitizing pollen such as alder, hazel, hornbeam, oak and chestnut because of the presence of PR10 allergens. PR10 allergens have indeed been described in other Quercus species. Objective Our goals were to determine the rate of sensitization to Q. ilex in central Spain and the associated frequency of PFAS; secondly to identify and clone the Q. ilex allergen PR10. Methods We included 224 allergic patients with respiratory symptoms to estimate the rate of sensitization. A skin prick test (SPT) and ImmunoCAP were performed. A total of 38 Q. ilex-sensitized patients were tested using Western blotting to determine the rate of Que i 1. Peptides from Que i 1 were analysed by MALDI-TOF/TOF and Orbitrap LC-MSMS. The Que i 1 sequence was first obtained from the Holm oak transcriptome then cloned and expressed in bacteria. Results 59.8% of pollen-allergic children were sensitized to Q. ilex. We described and cloned the Q. ilex PR10, Que i 1, which has a sensitization rate of 60.5% and was recognized by 65.4% patients reporting PFAS. Conclusion and clinical relevance Sensitization to Q. ilex pollen has increased significantly since 1995. This sensitization could be important, as the presence of PFAS in this population is higher than in patients not sensitized to Q. ilex. The first Q. ilex allergen has been described and is related to PFAS in Spanish patients sensitized to PR10 but not exposed to birch pollen.

The effect and responses to drought stress were analyzed in Quercus ilex L. seedlings using a nontargeted metabolomic approach, implementing the approaches of previous studies in which other -omics platforms, transcriptomics, and proteomics were employed. This work aimed to characterize the Q. ilex leaf metabolome, determining possible mechanisms and molecular markers of drought tolerance and identifying putative bioactive compounds. Six-month-old seedling leaves subjected to drought stress imposed by water withholding under high-temperature and irradiance conditions were collected when leaf fluorescence decreased by 20% (day 17) and 45% (day 24) relative to irrigated seedlings. A total of 3934 compounds were resolved, with 616 being variable and 342 identified, which belonged to five chemical families. Out of the identified compounds, 33 were variable, mostly corresponding to amino acids, carboxylic acids, benzenoids, flavonoids and isoprenoids. Epigallocatechin, ellagic acid, pulegone, indole-3-acrylic acid and dihydrozeatin-O-glucoside were up-accumulated under drought conditions at both sampling times. An integrated multi-omics analysis of phenolic compounds and related enzymes was performed, revealing that some enzymes involved in the flavonoid pathways (chalcone synthase, anthocyanidin synthase and anthocyanidin reductase) were up-accumulated at day 24 in non-irrigated seedlings. Some putative markers of tolerance to drought in Q. ilex are proposed for assisting breeding programs based on the selection of elite genotypes.

A província de Córdoba concentra 88% da área de cultivo de amendoim (Arachis hypogea L.) na Argentina. O uso de rizobactérias promotoras de crescimento de plantas (PGPR) é uma alternativa para reduzir os suprimentos sintéticos e melhorar o rendimento das colheitas. Pseudomonas e Bacillus são os principais gêneros que induzem o crescimento da cultura quando associados í rizosfera. O objetivo deste trabalho foi avaliar o impacto do PGPR em alguns traços associados ao crescimento, tanto em condições de campo como de câmara. O PGPR estimulava o crescimento das raízes, a biomassa total e o rendimento das culturas de amendoim no campo. O crescimento radicular estava entre +10 e +150% em relação ao controle. O PGPR aumentou o rendimento entre 26-44% em relação ao controle. Os resultados sugerem efeitos positivos do PGPR sobre a cultura do amendoim. A província de Córdoba concentra 88% da área de cultivo de amendoim (Arachis hypogea L.) na Argentina. O uso de rizobactérias promotoras de crescimento de plantas (PGPR) é uma alternativa para reduzir os suprimentos sintéticos e melhorar o rendimento das colheitas. Pseudomonas e Bacillus são os principais gêneros que induzem o crescimento da cultura quando associados í rizosfera. O objetivo deste trabalho foi avaliar o impacto do PGPR em alguns traços associados ao crescimento, tanto em condições de campo como de câmara. O PGPR estimulava o crescimento das raízes, a biomassa total e o rendimento das culturas de amendoim no campo. O crescimento radicular estava entre +10 e +150% em relação ao controle. O PGPR aumentou o rendimento entre 26-44% em relação ao controle. Os resultados sugerem efeitos positivos do PGPR sobre a cultura do amendoim. Córdoba province accounts fo r 88% of peanut (Arachis hypogea L.) grown area in Argentina. The use of plant growth-promoting rhizobacteria (PGPR) is an alternative to reduce synthetic supplies and improve crop yields. Pseudomonas and Bacillus are the main crop growth-inducing genera when associated with the rhizosphere. The objective of this work was to evaluate the impact of PGPR on some traits associated with growth, both under field and chamber conditions. PGPR stimulated root growth, total biomass, and yield of peanut crops in the field. Root growth was between +10 and +150% relative to the control. PGPR increased yield between 26-44% over the control. The results suggest positive effects of PGPR on the peanut crop.

Fusarium circinatum, causing pine pitch canker (PPC), affects conifers productivity and health worldwide. Selection and breeding for resistance arises as the most promising approach to fight PPC. Therefore, it is crucial to explore the response of hosts with varying levels of susceptibility to PPC to unveil the genes/pathways behind these phenotypes. We evaluated the dynamics of the needle proteome of a susceptible (Pinus radiata) and a relatively resistant (Pinus pinea) species upon F. circinatum inoculation by GeLC-MS/MS. Integration with physiological data and validation of key genes by qPCR allowed to identify core pathways regulating these contrasting responses. In P. radiata, the pathogen may target both the secondary metabolism to negatively regulate immune response and chloroplast redox proteins to increase energy-producing pathways for amino acid production in its favour. In contrast, chloroplast redox regulation may assure redox homeostasis in P. pinea, as well as nonenzymatic antioxidants. The presence of membrane trafficking-related proteins exclusively in P. pinea likely explains its defence response against F. circinatum. A crosstalk between abscisic acid and epigenetic regulation of gene expression is also proposed in PPC response. These results are useful to support breeding programs aiming to achieve PPC resistance.

Drought is one of the main causes of mortality in holm oak (Quercus ilex) seedlings used in reforestation programs. Although this species shows high adaptability to the extreme climate conditions prevailing in Southern Spain, its intrinsic genetic variability may play a role in the differential response of some populations and individuals. The aim of this work was to identify proteins and derived proteotypic peptides potentially useful as putative markers for drought tolerance in holm oak by using a targeted post-acquisition proteomics approach. For this purpose, we used a set of proteins identified by shotgun (LC-MSMS) analysis in a drought experiment on Q. ilex seedlings from four different provenances (viz. the Andalusian provinces Granada, Huelva, Cadiz and Seville). A double strategy involving the quantification of proteins and target peptides by shotgun analysis and post-acquisition data analysis based on proteotypic peptides was used. To this end, an initial list of proteotypic peptides from proteins highly represented under drought conditions was compiled that was used in combination with the raw files from the shotgun experiment to quantify the relative abundance of the fragment’s ion peaks with the software Skyline. The most abundant peptides under drought conditions in at least two populations were selected as putative markers of drought tolerance. A total of 30 proteins and 46 derived peptides belonging to the redox, stress-related, synthesis,-folding and degradation, and primary and secondary metabolism functional groups were thus identified. Two proteins (viz., subtilisin and chaperone GrpE protein) were found at increased levels in three populations, which make them especially interesting for validation drought tolerance markers in subsequent experiments. Fil: San Eufrasio, Bonoso. Universidad de Córdoba; España Fil: Bigatton, Ezequiel Darío. Universidad de Córdoba; España. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias. Departamento de Recursos Naturales. Cátedra de Microbiología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Guerrero Sánchez, Victor M.. Universidad de Córdoba; España Fil: Chaturvedi, Palak. Vienna University of Technology; Austria Fil: Jorrín Novo, Jesús V.. Universidad de Córdoba; España Fil: Rey, María Dolores. Universidad de Córdoba; España Fil: Castillejo Sánchez, María Ángeles. Universidad de Córdoba; España

Advances in proteomic equipment, algorithms and wet protocols are being increasingly reported. Each step in the experimental workflow must be adapted and optimized to the target experimental system and objectives. The influence of the amount of peptides loaded onto the column in shotgun platforms has rarely been considered to date even though it dictates the confidence with which proteins can be identified and quantified. An experiment using variable dilutions of protein equivalent mixtures of root, leaf and seed tissue extracts of Quercus ilex was performed by subjecting BSA protein equivalent amounts of 1–100 μg to SDS-PAGE, the resulting bands being trypsin digested and peptides (10–1000 ng protein equivalents) loaded onto an LC column. Mass spectra were used to identify proteins against the in-house Q. ilex transcriptome database. Determinations included SEQUEST quantification (average of the three most abundant distinct peptides for each protein) and proteotypic peptides. The number of proteins identified was found to depend on peptide load and to peak at 2054 with 600 ng. Smaller loads led to linearly decreasing identifications from 1859 with 400 ng to 495 with 10 ng. Both quantification strategies provided similar results. The linear dynamic range was from 100 to 600 ng.

A força da peg é uma característica importante da planta para evitar perdas na colheita. Este atributo é geralmente medido exercendo uma força mecânica através de um manómetro electrónico ou célula de carga de tensão até que ocorra o desprendimento da peg. O desenvolvimento de plataformas electrónicas como o Arduino®, que possuem hardware e software livre e de código aberto, permitiria obter medições de força da peg de forma rápida e barata. O objetivo deste trabalho era desenvolver um sistema electrónico económico de aquisição de dados baseado no Arduino® para medir a força da peg. Para o desenvolvimento de protótipos de hardware e software foi necessário criar o medidor de força da peg. A calibração foi avaliada através de R2, erro quadrático médio (RMSE), e erro de desvio médio (MBE). Os resultados foram 0,999, 0,014 g, e -0,003 g para R2, RMSE, e MBE, respectivamente. O medidor de força da peg foi capaz de detectar diferenças na força da peg entre tratamentos. Por conseguinte, este dispositivo é uma ferramenta importante para identificar melhores datas de colheita. La fuerza de los hilos que sujetan las vainas es un rasgo importante de la planta para evitar pérdidas durante el arrancado del cultivo, pero puede variar debido a prácticas agronómicas y características del cultivo. Este atributo se suele medir ejerciendo una fuerza mecánica a través de un medidor de fuerza electrónico o una célula de carga de tensión hasta que se produce el desprendimiento de la pica. El desarrollo de plataformas electrónicas como Arduino®, que disponen de hardware y software libre y gratuito, permitiría obtener mediciones de la fuerza de los hilos de las vainas de forma rápida y económica. El objetivo de este trabajo fue desarrollar un sistema económico de obtención de datos electrónicos basado en Arduino para medir la fuerza de las vainas. Para crear el medidor de fuerza de vainas fue necesario desarrollar prototipos de hardware y software. La calibración se evaluó mediante el R2, el error cuadrático medio (RMSE) y el error de sesgo medio (MBE). Los resultados fueron de 0,999, 0,014 g y -0,003 g para R2, RMSE y MBE, respectivamente. Por lo tanto, este dispositivo es una herramienta importante para identificar las fechas óptimas de arrancado del maní. Peg strength is an important plant trait to avoid digging losses although it can vary due to agronomic practices and cultivar characteristics. This attribute is usually measured by exerting a mechanical force through an electronic force gauge or tension load cell until peg detachment occurs. The development of electronic platforms such as Arduino®, which have free and open-source hardware and software would allow obtaining peg strength measurements quickly and inexpensively. The goal of this work was to develop an economic electronic data acquisition system Arduino-based for measuring peg strength. To create the peg strength meter was necessary to develop hardware and software prototypes. The calibration was assessed through R2, root mean square error (RMSE), and mean bias error (MBE). The results were 0.999, 0.014 g, and -0.003 g for R2, RMSE, and MBE, respectively. The Peg Strength meter was able to detect differences in peg strength between treatments. Therefore, this device is an important tool to identify appropriate digging dates.

Analyzing differences in tolerance to drought in Quercus spp., and the characterization of these responses at the species and individual population level, are imperative for the selection of resilient elite genotypes in reforestation programs. The main objective of this work was to evaluate differences in the response and tolerance to water shortage under in five Quercus spp. and five Andalusian Q. ilex populations at the inter- and intraspecies level. Six-month-old seedlings grown in perlite were subjected to drought treatments by withholding water for 28 days under mean 37 &deg, C temperature, 28 W m-2 solar irradiance, and 41% humidity. The use of perlite as the substrate enabled the establishment of severe drought stress with reduction in water availability from 73% (field capacity) to 28% (dryness), corresponding to matric potentials of 0 and &minus, 30 kPa. Damage symptoms, mortality rate, leaf water content, photosynthetic, and biochemical parameters (amino acids, sugars, phenolics, and pigments) were determined. At the phenotypic level, based on damage symptoms and mortality, Q. ilex behaved as the most drought tolerant species. Drought caused a significant decrease in leaf fluorescence, photosynthesis rate, and stomatal conductance in all Quercus spp. analyzed, being less pronounced in Q. ilex. There were not differences between irrigated and non-irrigated Q. ilex seedlings in the content of sugar and photosynthetic pigments, while the total amino acid and phenolic content significantly increased under drought conditions. As a response to drought, living Q. ilex seedlings adjust stomata opening and gas exchange, and keep hydrated, photosynthetically active, and metabolically competent. At the population level, based on damage symptoms, mortality, and physiological parameters, the eastern Andalusian populations were more tolerant than the western ones. These observations inform the basis for the selection of resilient genotypes to be used in breeding and reforestation programs.

Quercus ilex is the dominant tree species in natural forest ecosystems across the Mediterranean Basin and in the agrosilvopastoral system dehesa, which has a high ecological and economical significance. As in other forestry species, survival in Q. ilex is threatened by long periods of drought. This paper reports the transcriptome and proteome profiles of 6-month-old seedlings subjected to severe drought conditions. Drought was imposed by water withholding in seedlings grown in perlite for 28 days. Seedling leaves were collected when leaf fluorescence had decreased by 20% and 45% relative to well-watered seedlings. The transcriptome and proteome were analyzed by using Illumina and shotgun platforms. The quality and confidence of the mRNA and protein identifications and quantifications were assessed, obtaining 25,169 transcripts and 3312 proteins. Variable transcripts and proteins were analyzed by Venn diagram, Pearson's correlation, GO enrichment, KEGG pathways, multivariate analysis and interaction networks. Despite the poor correlation between mRNA and protein, both platforms gave a complementary view of the changes in the abundance of several gene products under drought conditions and indicated that gene expression regulation and translation to phenotype is quite complex and gene-specific. As a general tendency, while transcripts and proteins of the metabolism were down-accumulated, those of stress related were up-accumulated. Out of the variable dataset, four gene products (viz., FtSH6, CLPB1, CLPB3, and HSP22) were up-accumulated at both omics levels at the two surveyed times, being the first work where they are described in drought response in forest species. These chaperones and proteases could be considered as potential drought tolerance markers to be used in the selection of elite, resilient genotypes, and in breeding programs.

Gel electrophoresis-based and shotgun approaches are the most employed proteomic platforms in plant biology research, with the latter replacing the former in the last years. We have compared 2-DE-MALDI-TOF/TOF and GeLC-Orbitrap/MS analyses using the same protein extracts from Quercus ilex cotyledons at different development stages. The results obtained (ProteomeXchange available data, PXD020603) showed that both platforms were complementary, showing common and specific proteins identified in each case, but leading to similar biological conclusions. Protein analysis identified 562 spots in gel-based (292 variables) and 2409 proteins in shotgun (560 variables), that were detected with both platforms and represent common key pathways related to maturation and germination. The main differences concern hormone metabolism, storage and late embryogenesis abundant proteins. Deeper proteome coverage was obtained with the shotgun approach, with a greater number of metabolic pathways represented, as gibberellin biosynthesis, not observed in the gel-based analysis. Nevertheless, several storage proteins, highly abundant in cotyledons and well represented in gel-based platform were not identified using the shotgun platform. These results support that when analyzing any plant biological process, the use of both platforms is complementary rather than redundant, that favors an in-depth proteomic analysis and a more confident biological interpretation of the data obtained.

This research was funded by the Ministerio de Economía y Competitividad -Programa Estatal de I+D+i Orientada a los Retos de la Sociedad (AGL2009-12243-C02-02; BIO2015-64737-R)., Rey, M.-D., Castillejo, M.Á., Sánchez-Lucas, R., Guerrero-Sanchez, V.M., López-Hidalgo, C., Romero-Rodríguez, C., Valero-Galván, J., Sghaier-Hammami, B., Simova-Stoilova, L., Echevarría-Zomeño, S., Jorge, I., Gómez-Gálvez, I., Papa, M.E., Carvalho, K., Rodríguez De Francisco, L.E., Maldonado-Alconada, A.M., Valledor, L., Jorrín-Novo, J.V.

While reviewing the plant proteomics topic, both from a conceptual and methodological point of view and with a historical perspective, recent advances, current states, challenges, and future directions of the field are discussed. Proteomics is moving, even though very slowly, from the descriptive era to a new era in which data start to be validated and integrated with other classic and -omics approaches, in the Systems Biology direction. This review is organized in different sections, starting with a general and historical introduction, moving to platforms and techniques employed currently, data validation to confidently conclude from a biological point of view, workflow in a multi-omics experiment, highlighting some illustrative investigations, and finishing with some conclusions and remarks. As quite reviews have been already published in the field of mass spectrometry-based proteomics and to avoid being repetitive, this review only reports the most relevant contributions published recently by our research groups and those found in the literature.

By using two complementary proteomics, gel-based and gel-free (shotgun) approaches, the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth have been compared. Proteins were extracted from embryo axis, radicle and shoot tissues at different developmental stages. Proteins were subjected to one- and two-dimensional gel electrophoresis. A multivariate analysis (PCA) revealed that SDS-PAGE clearly separated germination (0–24 h post-imbibition), postgermination (72–216 h post-imbibition) and early seedling growth stages (2 weeks post-imbibition). Image analysis of the two-dimensional gels revealed a total of 732 spots, 103 of which were significantly variable among developmental stages. After MALDI-TOF/TOF MS analysis, 90 spots were identified, belonging to six main functional categories: carbohydrate, amino acids, energy, and protein metabolism, biosynthesis of secondary metabolites, and redox processes. The gel-based approach disclosed important metabolic changes that occurred in the holm oak seed after the germination. However, few proteins were significantly altered during the germination period (from 0 h to 24 h post imbibition) and, because of that, a further shotgun analysis was therefore used to analyse changes in the protein profile during seed germination. Up to 1250 proteins could be confidently identified, with 153 being variable. They belonged to the main functional categories of carbohydrate, amino acids and secondary metabolism, protein degradation, and responses to abiotic stress. The accumulation of proteases and amino acids metabolism proteins in mature seeds can be reflecting the production of energy from the mobilization of storage proteins to start germination. These results, therefore, corroborate the hypothesis that the mature non-orthodox seeds of Q. ilex have all the machinery necessary for rapidly resuming metabolic activities and starting the germination process, in contrast to that occurs in orthodox seeds, which metabolic activity ceases in mature dry seeds. The use of a genus-specific database combined with the public Viridiplantae database improved the quality and quantity of protein identification in this orphan species. In addition, both proteomics approaches (gel-based and shotgun) were complementary, with shotgun increasing by over two-fold the coverage of the proteome analysed. Both approaches provided similar results and supported the same conclusions on the metabolic switch experienced by the seed upon germination. Significance The optimal seed germination is a prerequisite for successful seedling establishment and plant vigour, being of great relevance in the case of crops and commercial woody plants. By using a complementary gel-based and gel-free proteomic strategy we have study the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth. The contribution of this work is of great importance, due to the complemented proteomic approaches giving similar clues to the metabolic state of the mature Q. ilex seed before the germination starts, and the metabolic switch experienced by the imbibed acorn until the seedling is established.

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

Yield stability of legume crops is constrained by a number of pest and diseases. Major diseases are rusts, powdery and downy mildews, ascochyta blights, botrytis gray molds, anthracnoses, damping-off, root rots, collar rots, vascular wilts and white molds. Parasitic weeds, viruses, bacteria, nematodes and damages caused by chewing and sap-sucking insects add to this long list of constraints for legume production. Their incidence and relative importance together with current understanding of their interactions with the host plants are presented. State of the art of current achievements and limitations for breeding for biotic stress resistance are listed and critically discussed. The recent development of large scale phenotyping, genome sequencing and analysis of gene, protein and metabolite expressions can be of great help to further decipher plant-pathogen interactions and identify key resistance components that may be introgressed into crop plants through breeding.

Proteomics analysis has been performed in leaf tissue from field date palm trees showing the brittle leaf disease (BLD) or maladie des feuilles cassantes, the main causal agent of the date palm decline in south Tunisia. To study the evolution of the disease, proteins from healthy and affected leaves taken at three disease stages (S1, S2 and S3) were trichloroacetic acid acetone extracted and subjected to two-dimensional gel electrophoresis (5-8 pH range). Statistical analysis showed that the protein abundance profile is different enough to differentiate the affected leaves from the healthy ones. Fifty-eight variable spots were successfully identified by matrix-assisted laser desorption ionization time of flight, 60 % of which corresponded to chloroplastic ones being involved in the photosynthesis electronic chain and ATP synthesis, metabolic pathways implicated in the balance of the energy, and proteases. Changes in the proteome start at early disease stage (S1), and are greatest at S2. In addition to the degradation of the ribulose-1.5-bisphosphate carboxylase oxygenase in affected leaflets, proteins belonging to the photosynthesis electronic chain and ATP synthesis decreased following the disease, reinforcing the relationship between BLD and manganese deficiency. The manganese-stabilizing proteins 33 kDa, identified in the present work, can be considered as protein biomarkers of the disease, especially at early disease step.

Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

Fusarium oxysporum f. sp. pisi (Fop) is an important and destructive pathogen affecting pea crop (Pisum sativum) throughout the world. Control of this disease is achieved mainly by integration of different disease management procedures. However, the constant evolution of the pathogen drives the necessity to broaden the molecular basis of resistance to Fop. Our proteomic study was performed on pea with the aim of identifying proteins involved in different resistance mechanisms operating during F. oxysporum infection. For such purpose, we used a two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) analysis to study the root proteome of three pea genotypes showing different resistance response to Fop race 2. Multivariate statistical analysis identified 132 differential protein spots under the experimental conditions (genotypes/treatments). All of these protein spots were subjected to mass spectrometry analysis to deduce their possible functions. A total of 53 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. The following main functional categories were assigned to the identified proteins: carbohydrate and energy metabolism, nucleotides and aminoacid metabolism, signal transduction and cellular process, folding and degradation, redox and homeostasis, defense, biosynthetic process and transcription/translation. Results obtained in this work suggest that the most susceptible genotypes have increased levels of enzymes involved in the production of reducing power which could then be used as cofactor for enzymes of the redox reactions. This is in concordance with the fact that a ROS burst occurred in the same genotypes, as well as an increase of PR proteins. Conversely, in the resistant genotype proteins responsible to induce changes in the membrane and cell wall composition related to reinforcement were identified. Results are discussed in terms of the differential response to Fop., The authors acknowledge the Spanish Ministry of Science and Innovation for funding of this work (AGL2011-22524) co-financed by FEDER. Ma Angeles Castillejo was funded by a postdoctoral fellowship from the Spanish Ministry of Education, through the Mobility Program R-D + I 2008–2011.

Orobanche and Phelipanche species are root parasitic plants some of which infect important crops, causing extensive damage. In an attempt to unravel molecular systematic in these genera using protein markers, we established the protein profile of twelve species by two-dimensional gel electrophoresis (2-DE). Proteins were detected by Sypro Ruby staining and the gel images were digitalised and analysed. The 2-DE maps contained from 30 to 140 protein spots for the analysed species. A virtual master gel was built with spots present in all species and revealing the presence of 260 protein spots. Only one protein was monomorphic, while 143 were unique to a single species. Proteins were scored for the presence or absence of homologous spots to create a binary matrix. Phylogenetic analysis was carried out using maximum parsimony and distance method. The trees obtained were rooted using Cistanche phelypaea as an out-group. Both analyses showed Orobanche as a monophyletic group. AMOVA analysis revealed phenotypic diversity between species within genera, as well as a considerable level of variation between genera. This is a first step to help in the phylogenetic classification of parasitic plants by means of proteomics.