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Pinus occidentalis Swartz is the dominant species of forest ecosystems in the Dominican Republic, located between 200 and 3000 m above sea level, with extensive and overexploited natural populations. However, over the years, various restoration plans have been performed, which could affect the genetic structure of P. occidentalis. Despite being the species with the highest occurrence in the Dominican forests, there is no existing information on the genetic structure and molecular characterization among natural populations with limited information on both phenological and productive characterization. In this study, the genetic structure, diversity, and gene flow of the five P. occidentalis natural populations of the Dominican Republic were determined using microsatellite markers. A total of 145 individuals were genotyped with eight polymorphic chloroplastic microsatellites, producing an average of 41 haplotypes with high genetic diversity across populations (HE = 0.90). Significant population genetic structure was found between populations (FST = 0.123). These results reflect the impact of reforestation programs on natural populations and diluting the natural genetic signature. Analysis of population genetic data are, therefore, crucial for the breeding and conservation programs of P. occidentalis in the country.

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.

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.

Cakile maritima is a halophytic plant model that is well known by its ability to tolerate high salt concentrations. Salinity was reported to improve the tolerance of halophytes to several abiotic stresses; however, the involvement of salt in the tolerance to biotic stress is still scant. In the present work, the effect of salt on C. maritima responses towards the pathogenic Alternaria alternata was investigated. For that, C. maritima seeds were germinated for four weeks. Plants were then divided into four groups: i) Plants irrigated with salt (200mM NaCl); ii) Plants infested by fungus; iii) Plants irrigated with salt and infested by fungus and finally control plants (0mM NaCl, without inoculation). Our results showed that upon salt stress or fungal attack, plants reduced biomass production, hydration status and photosynthetic performance which were associated with a decrease in the gas exchange and chlorophyll fluorescence parameters, with a more pronounced effect upon fungal attack. However, under combined stress, a significant increase of these parameters was noticed, with a level close to that of control. Concerning nutrient contents, K, Zn, Fe, Cu and Mg decreased in the C. maritima leaves exposed to both stresses applied individually. In contrast, all these nutrients were increased in plants grown under combined stress. Taken together, we can conclude that plants grown under combined stresses had better growth rate and physiological performance compared to all other treated plants, and that salt may be the key in improving the C. maritima ability to tolerate fungal attack.

The date palm, a central plant in the fragile oasis ecosystem, is considered one of the fruit species most tolerant to salt stress. However, the tolerance mechanisms involved are yet to be addressed and their evaluation until now was mainly based on heterogenous plant material such as seedlings or limited to in vitro experiment conditions. For these reasons, we propose to deepen our knowledge of the morphological and physiological responses to salt stress using acclimated ex vitro plants resulting from the propagation of a single genotype. The plants were irrigated with 0, 150, 300, or 450 mM NaCl solutions for four months. Our results showed that the influence of water salinity on growth and ion-homeostasis regulation was very dependent on stress levels. The 150 mM NaCl concentration was found to improve dry biomass by about 35%, but at higher salt concentrations (300 and 450 mM) it decreased by 40–65%. The shoot:root dry mass ratio decreased significantly at the 150 mM NaCl water concentration and then increased with increasing water salt concentration. The leaf:root ratio for Na+ and Cl− decreased significantly with increasing water salinity up to a concentration of 300 mM NaCl, and then stabilized with similar values for 300 mM and 450 mM NaCl. In contrast to Na+ and Cl−, leaf K+ content was significantly higher in the leaf than in the root for all salt treatments. Unlike Na+ and K+, Cl− was expelled to the surface of leaves in response to increased water salinity. Overall, date palm plants appear to be more capable of excluding Cl− than Na+ and of changing biomass allocation according to salt-stress level, and their leaves and roots both appear to play an important role in this tolerance strategy., All authors are funded through the Small Research group project from the Deanship of Scientific Research at King Khalid University under research grant number (R.G.P.1/295/43).

This study aimed to evaluate a complete nutritional composition in the seeds Quercus virginiana to compare this nutritional composition with three Mediterranean Quercus species. We analyzed the seed morphometry, proximate composition, phytochemicals, and antioxidant capacity. The seed of Q. virginiana presented the smaller seed size and weight, while Q. suber presented the highest values. Moreover, Q. virginiana seeds showed the highest amounts of sugar and total lipids, digestibility, energy, palmitic acid, and stearic acid. On the other hand, Q. virginiana seeds showed the lowest values of linoleic acid. Moreover, Q. coccifera seeds presented the highest total phenolics and flavonoids contents and antioxidant activity. The clustering analysis revealed a significant similarity in seed morphometry and nutritional composition between the Mediterranean Q. ilex and Q. suber, grouping with the American Q. virginiana, but to a considerable distance, by contrast, the Mediterranean Q. coccifera was the most distant in the clustering analysis. The content of phenolics and flavonoids and digestibility value were the variables that contributed to the separation to a greater extent in the clustering of the four species. The nutritional and biological activity assessment of plant seed may be considered as an essential mission to find new sustainable sources and novel chemical agents. In this sense, Quercus seeds may be an alternative and a competitive food source for the agri-food industry.

TheJuniperusgenus has long been used and studied for the chemical components of its aerial parts (leaves, bark, twigs) and their bioactivity. Nevertheless, these studies and their compilation have been primarily focused on Europe and North America distributed taxa, leaving the knowledge and economic potential of the endangered Caribbean taxa highly underrepresented in literature. Although, these conifers have been barely investigated for their bioactive compounds, bibliography does indicate the presence of potent antitumoral, anti-inflammatory and antimicrobial molecules such as deoxypodophyllotoxin, podophyllotoxin, amentoflavone and widdrol. Additional phytochemical potential can also be inferred from the systematical essential oil studies of the taxa, the only source of chemical composition information on most of them. These investigations can aid in the narrowing down of the possible bioactivities their lipidic extracts may possess, while also providing clues for the bioassays necessary to confirm them. This review aims to compile the known information on the usage, bioactivity and chemical composition of the HispaniolianJ. graciliorvarieties and their phylogenetically proximal taxa (J. graciliorvar.saxicola,J. barbadensisandJ. bermudiana), to propitiate more holistic and in depth chemical studies on these potential phytochemical sources, in turn providing an economical incentive for their conservation., El géneroJuniperusha sido utilizado y estudiado a lo largo de los años por la bioactividad de los compuestos fitoquímicos encontrados en sus tejidos (hojas, corteza, ramas). Mayoritariamente, dichas investigaciones y las recopilaciones de estas se han centrado en taxones distribuidos en Europa y América del Norte, dejando de lado la investigación y potencial económico de otros como los caribeños, que actualmente se encuentran en peligro de extinción. Aunque los compuestos bioactivos de los últimos apenas han sido estudiados, en la literatura se reportan potentes moléculas antitumorales, antiinflamatorias y antimicrobianas como la desoxipodofilotoxina, podofilotoxina, amentoflavona y el widdrol en su composición. De igual manera, investigaciones fitoquímicas realizadas a sus aceites esenciales con objetivo taxonómico, que en muchos casos representan la única fuente de información química de estosJuniperusdel Caribe, permiten inferir propiedades bioactivas adicionales de los mismos, lo que resalta su posible potencial. Este trabajo tuvo como objetivo recopilar la información conocida sobre el uso, la bioactividad y la composición química de las variedades deJ. graciliorendémicas de la Hispaniola y sus taxones filogenéticamente cercanos (J. graciliorvar.saxicola,J. barbadensisyJ. bermudiana), para propiciar futuros estudios holísticos y exhaustivos de los metabolitos encontrados en estas posibles fuentes de compuestos fitoquímicos, proporcionando así un incentivo económico para su conservación.

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

The genus Quercus (oak), family Fagaceae, comprises around 500 species, being one of the most important and dominant woody angiosperms in the Northern Hemisphere. Nowadays, it is threatened by environmental cues, which are either of biotic or abiotic origin. This causes tree decline, dieback, and deforestation, which can worsen in a climate change scenario. In the 21st century, biotechnology should take a pivotal role in facing this problem and proposing sustainable management and conservation strategies for forests. As a non-domesticated, long-lived species, the only plausible approach for tree breeding is exploiting the natural diversity present in this species and the selection of elite, more resilient genotypes, based on molecular markers. In this direction, it is important to investigate the molecular mechanisms of the tolerance or resistance to stresses, and the identification of genes, gene products, and metabolites related to this phenotype. This research is being performed by using classical biochemistry or the most recent omics (genomics, epigenomics, transcriptomics, proteomics, and metabolomics) approaches, which should be integrated with other physiological and morphological techniques in the Systems Biology direction. This review is focused on the current state-of-the-art of such approaches for describing and integrating the latest knowledge on biotic and abiotic stress responses in Quercus spp., with special reference to Quercus ilex, the system on which the authors have been working for the last 15 years. While biotic stress factors mainly include fungi and insects such as Phytophthora cinnamomi, Cerambyx welensii, and Operophtera brumata, abiotic stress factors include salinity, drought, waterlogging, soil pollutants, cold, heat, carbon dioxide, ozone, and ultraviolet radiation. The review is structured following the Central Dogma of Molecular Biology and the omic cascade, from DNA (genomics, epigenomics, and DNA-based markers) to metabolites (metabolomics), through mRNA (transcriptomics) and proteins (proteomics). An integrated view of the different approaches, challenges, and future directions is critically discussed.

Date palm (Phoenix dactylifera L.), a dioecious species, represents a major agricultural component of arid and semiarid regions. It plays a fundamental role in the socioeconomic balance. Dates, indeed, represent a major income and food source for local populations in the Middle East and North Africa. A better understanding of the plant’s response to biotic and abiotic stress may help in improving date production, especially of elite cultivars. For this reason, molecular tools including genomics, transcriptomics and proteomics were developed to realize this response. Recently, proteomics has become widely used in biological research to explain several unknown biological functions. In the date palm production field, it is being used to elucidate the mechanism of plant resistance to environmental stress. The present chapter describes proteomic approaches used to compare the proteome of zygotic and somatic date palm embryos. Additionally, an explanation is given about the effect of the addition of different supplements to medium culture on the protein content and profile of somatic embryos. Furthermore, the response of date palm to abiotic stress including brittle leaf disease, salinity and drought at proteomic levels is described and discussed.

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.

Unlike orthodox species, seed recalcitrance is poorly understood, especially at the molecular level. In this regard, seed maturation and germination were studied in the non-orthodox Quercus ilex by using a proteomics strategy based on two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization/time of flight (2-DE-MALDI-TOF).Cotyledons and embryo/radicle were sampled at different developmental stages, including early (M1–M3), middle (M4–M7), and late (M8–M9) seed maturation, and early (G1–G3) and late (G4–G5) germination. Samples corresponding to non-germinating, inviable, seeds were also included. Protein extracts were subjected to 2-dimensional gel electrophoresis (2-DE) and changes in the protein profiles were analyzed. Identified variable proteins were grouped according to their function, being the energy, carbohydrate, lipid, and amino acid metabolisms, together with protein fate, redox homeostasis, and response to stress are the most represented groups. Beyond the visual aspect, morphometry, weight, and water content, each stage had a specific protein signature. Clear tendencies for the different protein groups throughout the maturation and germination stages were observed for, respectively, cotyledon and the embryo axis. Proteins related to metabolism, translation, legumins, proteases, proteasome, and those stress related were less abundant in non-germinating seeds, it related to the loss of viability. Cotyledons were enriched with reserve proteins and protein-degrading enzymes, while the embryo axis was enriched with proteins of cell defense and rescue, including heat-shock proteins (HSPs) and antioxidants. The peaks of enzyme proteins occurred at the middle stages (M6–M7) in cotyledons and at late ones (M8–M9) in the embryo axis. Unlike orthodox seeds, proteins associated with glycolysis, tricarboxylic acid cycle, carbohydrate, amino acid and lipid metabolism are present at high levels in the mature seed and were maintained throughout the germination stages. The lack of desiccation tolerance in Q. ilex seeds may be associated with the repression of some genes, late embryogenesis abundant proteins being one of the candidates.

Quercus ilex is a dominant tree species in the Mediterranean region with double economic and ecological importance and increasing use in reforestation. Seedling establishment is extremely vulnerable to environmental stresses, particularly drought. A time course study on physiological and proteomic response of holm oak to water limitation stress and recovery during early heterotrophic growth is reported. Applied stress led to diminution in plant water content and root growth, oxidative stress in roots and some alterations in the anti-oxidative protection. Plant parts differed substantially in soluble sugar and free phenolic content, and in their changes during stress and recovery. Proteomic response in holm oak roots and cotyledons was estimated using combined 1-DE/2-DE approach and protein identification by MALDI TOF-TOF PMF and MS/MS. A total of 127 differentially abundant protein species (DAPs) were identified. DAPs related to starch metabolism, lipid to sugar conversion, reserve proteins and their mobilization were typical for cotyledons. DAPs in roots were involved in sugar utilization, secondary metabolism and defense, including pathogenesis related proteins from PR-5 and PR-10 families. Results emphasize specific proteome signatures of separate plant parts as well as importance of sink-source interaction between root and cotyledon in the time course of stress and in recovery.

Lasiodiplodia theobromae is a phytopathogenic fungus that causes diseases not only in a broad number of plant hosts but also occasionally in humans. The capacity of L. theobromae to produce bioactive metabolites at 25 C (environmental mean temperature) and at 37 C (body mean temperature) was investigated. Two strains, CAA019 and CBS339.90, isolated respectively from a coconut tree and a human patient were characterized. The phytotoxicity and cytotoxicity (on mammalian cells) of the secretomes of both strains of L. theobromae were investigated. Also, phytotoxicity and cytotoxicity of pure compounds were evaluated. The phytotoxicity of the secretome of strain CAA019 was higher than the phytotoxicity of the secretome of strain CBS339.90 at 25 C. However, the phytotoxicity for both strains decreased when they were grown at 37 C. Only the secretome of strain CBS339.90 grown at 37 C induced up to 90% Vero and 3T3 cell mortality. This supports the presence of different metabolites in the secretome of strains CAA019 and CBS339.90. Metabolites typical of L. theobromae were isolated and identified from organic extracts of the secretome of both strains (e.g., 3-indolecarboxylic acid, jasmonic acid, lasiodiplodin, four substituted 2-dihydrofuranones, two melleins, and cyclo-(Trp-Ala)). Also, metabolites such as scytalone, not previously reported for this species, were isolated and identified. Metabolite production is affected by strain and temperature. In fact, some metabolites are strain specific (e.g., lasiodiplodin) and some metabolites are temperature specific (e.g., jasmonic acid). Although more strains should be characterized, it may be anticipated that temperature tuning of secondary-metabolite production emerges as a putative contributing factor in the modulation of L. theobromae pathogenicity towards plants, and also towards mammalian cells.

Substantial equivalence studies were performed in three Theobroma spp., cacao, bicolor and grandiflorum through chemical composition analysis and protein profiling of fruit (pulp juice and seeds). Principal component analysis of sugar, organic acid, and phenol content in pulp juice revealed equivalence among the three species, with differences in some of the compounds that may result in different organoleptic properties. Proteins were extracted from seeds and pulp juice, resolved by two dimensional electrophoresis and major spots subjected to mass spectrometry analysis and identification. The protein profile, as revealed by principal component analysis, was variable among the three species in both seed and pulp, with qualitative and quantitative differences in some of protein species. The functional grouping of the identified proteins correlated with the biological role of each organ. Some of the identified proteins are of interest, being minimally discussed, including vicilin, a protease inhibitor, and a flavonol synthase/flavanone 3-hydroxylase. Biological significance Theobroma grandiflorum and Theobroma bicolor are endemic Amazonian plants that are poorly traded at the local level. As close relatives of Theobroma cacao, they may provide a good alternative for human consumption and industrial purposes. In this regard, we performed equivalence studies by conducting a comparative biochemical and proteomics analysis of the fruit, pulp juice and seeds of these three species. The results indicated equivalent chemical compositions and variable protein profiles with some differences in the content of the specific compounds or protein species that may result in variable organoleptic properties between the species and can be exploited for traceability purposes.

The third edition of "Plant Proteomics Methods and Protocols," with the title "Advances in Proteomics Techniques, Data Validation, and Integration with Other Classic and -Omics Approaches in the Systems Biology Direction," was conceived as being based on the success of the previous editions, and the continuous advances and improvements in proteomic techniques, equipment, and bioinformatics tools, and their uses in basic and translational plant biology research that has occurred in the past 5 years (in round figures, of around 22,000 publications referenced in WoS, 2000 were devoted to plants).The monograph contains 29 chapters with detailed proteomics protocols commonly employed in plant biology research. They present recent advances at all workflow stages, starting from the laboratory (tissue and cell fractionation, protein extraction, depletion, purification, separation, MS analysis, quantification) and ending on the computer (algorithms for protein identification and quantification, bioinformatics tools for data analysis, databases and repositories). At the end of each chapter there are enough explanatory notes and comments to make the protocols easily applicable to other biological systems and/or studies, discussing limitations, artifacts, or pitfalls. For that reason, as with the previous editions, it would be especially useful for beginners or novices.Out of the 29 chapters, six are devoted to descriptive proteomics, with a special emphasis on subcellular protein profiling (Chapters 5 - 10 ), six to PTMs (Chapters 11 , and 14 - 18 ), three to protein interactions (Chapters 19 - 21 ), and two to specific proteins, peroxidases (Chapter 24 ) and proteases and protease inhibitors (Chapter 26 ). The book reflects the new trajectory in MS-based protein identification and quantification, moving from the classic gel-based approaches to the most recent labeling (Chapters 10 , 11 , 29 ), shotgun (Chapters 5 , 7 , 12 , 15 ), parallel reaction monitoring (Chapter 16 ), and targeted data acquisition (Chapter 13 ). MS imaging (Chapter 25 ), the only in vivo MS-based proteomics strategy, is far from being fully optimized and exploited in plant biology research. A confident protein identification and quantitation, especially in orphan species, of low-abundance proteins, is still a challenging task (Chapters 4 , 28 ).What is really new is the use of different techniques for proteomics data validation and their integration into other classic and -omics approaches in the systems biology direction. Chapter 2 reports on multiple extractions in a single experiment of the different biomolecules, nucleic acids, proteins, and metabolites. Chapter 27 describes how metabolic pathways can be reconstructed from multiple -omics data, and Chapter 3 network building. Finally, Chapters 22 and 23 deal with, respectively, the search for allele-specific proteins and proteogenomics.Around 200 groups were, almost 1 year ago, invited to take part in this edition. Unfortunately, only 10% of them kindly accepted. My gratitude to those who accepted our invitation but also to those who did not, as all of them have contributed to the plant proteomics field. I will enlist, in this introductory chapter, following my own judgment, some of the relevant papers published in the past 5 years, those that have shown us how to enhance and exploit the potential of proteomics in plant biology research, without aiming at giving a too exhaustive list.

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.

In the era of high-throughput biology, it is necessary to develop a simple pipeline for metabolic pathway reconstruction in plant orphan species. However, obtaining a global picture of the plant metabolism may be challenging, especially in nonmodel species. Moreover, the use of bioinformatics tools and statistical analyses is required. This chapter describes how to use different software and online tools for the reconstruction of metabolic pathways of plant species using existing pathway knowledge. In particular, Quercus ilex omics data is employed to develop the present pipeline.

Lasiodiplodia theobromae (Botryosphaeriaceae, Ascomycota) is a plant pathogen and human opportunist whose pathogenicity is modulated by temperature. The molecular effects of temperature on L. theobromae are mostly unknown, so we used a multi-omics approach to understand how temperature affects the molecular mechanisms of pathogenicity. The genome of L. theobromae LA-SOL3 was sequenced (Illumina MiSeq) and annotated. Furthermore, the transcriptome (Illumina TruSeq) and proteome (Orbitrap LC-MS/MS) of LA-SOL3 grown at 25 °C and 37 °C were analysed. Proteins related to pathogenicity (plant cell wall degradation, toxin synthesis, mitogen-activated kinases pathway and proteins involved in the velvet complex) were more abundant when the fungus grew at 25 °C. At 37 °C, proteins related to pathogenicity were less abundant than at 25 °C, while proteins related to cell wall organisation were more abundant. On the other hand, virulence factors involved in human pathogenesis, such as the SSD1 virulence protein, were expressed only at 37 °C. Taken together, our results showed that this species presents a typical phytopathogenic molecular profile that is compatible with a hemibiotrophic lifestyle. We showed that L. theobromae is equipped with the pathogenesis toolbox that enables it to infect not only plants but also animals.

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.

Water stress is of the major abiotic stress factors shifting the plant metabolism and subsequently plant growth, development and yield. In order to reduce the adverse impacts of water stress, herewith the study, the effects of salicylic acid priming on water stressed basil plants has been evaluated through physiological, biochemical and yield parameters. In this context, basil seeds (Ocimum basilicum) were initially primed with 0.05 and 0.1 mM concentration of salicylic acid (SA) and subsequently, basil plants were grown under well-watered and drought conditions. Accordingly, SA priming increased relative water content, water potential, quantum yield (Fv/Fm ratio), shoot height, stem diameter, dry weight, branch number, and leaf number. Furthermore, according to the regression analysis all variables were found to be significant by the t-test for contents of elements. However, interaction of water priming, field capacity and harvesting time was not significant regarding potassium content. According to the standardized regression coefficient (β), of the independent variables, field capacity was more influential factor on the all element accumulation but iron content and it was found to be at the first order in terms of relative importance on the content of elements and then followed by SA priming and harvest time. The content of elements, except Ca and Mg, were reduced with water stress but priming with SA increased the content of the elements. In addition, water stress and SA priming yielded higher contents of phenolic and flavonoid content coupled with higher antioxidant activities. Herewith the study, 1,8-cineol, linalool, eugenol and α-bergamotene were identified as the major essential oil components in all applications. However, treatments did not affect significantly their percentage.

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.

Quercus acorns have been used as a staple food since archeological times; currently, there is a renewed interest in the direct use of the acorns as nuts or as a source of consumable flour. In this regard, the phytochemical composition and variability of various morphotypes of acorns from Quercus ilex have been determined by using two platforms, near infrared spectroscopy (NIRS) and mass spectrometry (MS)-based metabolomics. According to NIRS, starch was the most abundant compound (50-60%). UHPLC-QToF analysis was used for untargeted metabolomic analysis and 192 metabolites were annotated. Principal component analysis (PCA) discriminated the morphotypes; 50 compounds out of 192 identified had the highest load over the first two PCA components (explaining 67.2% variability) and can be potential markers of variability. The data presented here support the value of acorns as a source of nutritionally rich compounds thus justifying their use a good alternative to other nuts.

Tomato chlorotic mottle virus (ToCMoV) is a widespread bipartite Begomovirus species found in tomato fields in Brazil. In this study, plant responses and putative mechanisms associated with the ‘Tyking’-derived recessive resistance to ToCMoV were investigated. Changes in the protein profile in the inoculated plants of two near isogenic tomato lines resistant (‘LAM 157’) and susceptible (‘Santa Clara’) to ToCMoV were analyzed. Seedlings were biolistically inoculated with an infectious ToCMoV clone. Leaves from infected plants (confirmed by PCR) were sampled at 15 days after inoculation. Proteins were extracted using phenol and analyzed by shotgun MS (2D-nanoUPLC/HDMSE). Out of the 534 identified proteins, 82 presented statistically significant differences in abundance, including 35 unique proteins displayed in the resistant tomato inoculated with ToCMoV. Proteins associated to chromatin structure, cytoskeleton structure, cuticle biosynthesis, and ubiquitin pathway were identified and their putative roles during virus infection process were discussed. The protein profile analysis allowed for the development of a hypothetical model showing how the resistant host cell responds to ToCMoV infection. The data obtained provide a better understanding of resistant mechanisms used by the host plant to contain viral infection and could be the basis for further investigation in other plant-begomovirus pathosystems. Biological significance In this study we propose a model of resistance to begomovirus in tomato and highlight host proteins, which could be targets for future investigations in plant-begomovirus pathosystems.

International audience; The present review is an update of the previous one published in Proteomics 2015 Reviews special issue [Jorrin-Novo, J. V. et al., Proteomics 2015, 15, 1089-1112] covering the July 2014-2015 period. It has been written on the bases of the publications that appeared in Proteomics journal during that period and the most relevant ones that have been published in other high-impact journals. Methodological advances and the contribution of the field to the knowledge of plant biology processes and its translation to agroforestry and environmental sectors will be discussed. This review has been organized in four blocks, with a starting general introduction (literature survey) followed by sections focusing on the methodology (in vitro, in vivo, wet, and dry), proteomics integration with other approaches (systems biology and proteogenomics), biological information, and knowledge (cell communication, receptors, and signaling), ending with a brief mention of some other biological and translational topics to which proteomics has made some contribution.

Advancements in high-throughput “Omics” techniques have revolutionized plant molecular biology research [...]

Unlike orthodox species, seed recalcitrance is poorly understood, especially at the molecular level. In this regard, seed maturation and germination were studied in the non-orthodox Quercus ilex by using a proteomics strategy based on two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization/time of flight (2-DE-MALDI-TOF).Cotyledons and embryo/radicle were sampled at different developmental stages, including early (M1&ndash, M3), middle (M4&ndash, M7), and late (M8&ndash, M9) seed maturation, and early (G1&ndash, G3) and late (G4&ndash, G5) germination. Samples corresponding to non-germinating, inviable, seeds were also included. Protein extracts were subjected to 2-dimensional gel electrophoresis (2-DE) and changes in the protein profiles were analyzed. Identified variable proteins were grouped according to their function, being the energy, carbohydrate, lipid, and amino acid metabolisms, together with protein fate, redox homeostasis, and response to stress are the most represented groups. Beyond the visual aspect, morphometry, weight, and water content, each stage had a specific protein signature. Clear tendencies for the different protein groups throughout the maturation and germination stages were observed for, respectively, cotyledon and the embryo axis. Proteins related to metabolism, translation, legumins, proteases, proteasome, and those stress related were less abundant in non-germinating seeds, it related to the loss of viability. Cotyledons were enriched with reserve proteins and protein-degrading enzymes, while the embryo axis was enriched with proteins of cell defense and rescue, including heat-shock proteins (HSPs) and antioxidants. The peaks of enzyme proteins occurred at the middle stages (M6&ndash, M7) in cotyledons and at late ones (M8&ndash, M9) in the embryo axis. Unlike orthodox seeds, proteins associated with glycolysis, tricarboxylic acid cycle, carbohydrate, amino acid and lipid metabolism are present at high levels in the mature seed and were maintained throughout the germination stages. The lack of desiccation tolerance in Q. ilex seeds may be associated with the repression of some genes, late embryogenesis abundant proteins being one of the candidates.

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.

Transcriptome analysis is widely used in plant biology research to explore gene expression across a large variety of biological contexts such as those related to environmental stress and plant-pathogen interaction. Currently, next generation sequencing platforms are used to obtain a high amount of raw data to build the transcriptome of any plant. Here, we compare Illumina and Ion Torrent sequencing platforms for the construction and analysis of the holm oak (Quercus ilex) transcriptome. Genomic analysis of this forest tree species is a major challenge considering its recalcitrant character and the absence of previous molecular studies. In this study, Quercus ilex raw sequencing reads were obtained from Illumina and Ion Torrent and assembled by three different algorithms, MIRA, RAY and TRINITY. A hybrid transcriptome combining both sequencing technologies was also obtained in this study. The RAY-hybrid assembly generated the most complete transcriptome (1,116 complete sequences of which 1,085 were single copy) with a E90N50 of 1,122 bp. The MIRAIllumina and TRINITY-Ion Torrent assemblies annotated the highest number of total transcripts (62,628 and 74,058 respectively). MIRA-Ion Torrent showed the highest number of shared sequences (84.8%) with the oak transcriptome. All the assembled transcripts from the hybrid transcriptome were annotated with gene ontology grouping them in terms of biological processes, molecular functions and cellular components. In addition, an in silico proteomic analysis was carried out using the translated assemblies as databases. Those from Ion Torrent showed more proteins compared to the Illumina and hybrid assemblies. This new generated transcriptome represents a valuable tool to conduct differential gene expression studies in response to biotic and abiotic stresses and to assist and validate the ongoing Q. ilex whole genome sequencing.

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.

Holm oak (Quercus ilex) is the most important and representative species of the Mediterranean forest and of the Spanish agrosilvo-pastoral “dehesa” ecosystem. Despite its environmental and economic interest, Holm oak is an orphan species whose biology is very little known, especially at the molecular level. In order to increase the knowledge on the chemical composition and metabolism of this tree species, the employment of a holistic and multi-omics approach, in the Systems Biology direction would be necessary. However, for orphan and recalcitrant plant species, specific analytical and bioinformatics tools have to be developed in order to obtain adequate quality and data-density before to coping with the study of its biology. By using a plant sample consisting of a pool generated by mixing equal amounts of homogenized tissue from acorn embryo, leaves, and roots, protocols for transcriptome (NGS-Illumina), proteome (shotgun LC-MS/MS), and metabolome (GC-MS) studies have been optimized. These analyses resulted in the identification of around 62629 transcripts, 2380 protein species, and 62 metabolites. Data are compared with those reported for model plant species, whose genome has been sequenced and is well annotated, including Arabidopsis, japonica rice, poplar, and eucalyptus. RNA and protein sequencing favored each other, increasing the number and confidence of the proteins identified and correcting erroneous RNA sequences. The integration of the large amount of data reported using bioinformatics tools allows the Holm oak metabolic network to be partially reconstructed: from the 127 metabolic pathways reported in KEGG pathway database, 123 metabolic pathways can be visualized when using the described methodology. They included: carbohydrate and energy metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, and biosynthesis of secondary metabolites. The TCA cycle was the pathway most represented with 5 out of 10 metabolites, 6 out of 8 protein enzymes, and 8 out of 8 enzyme transcripts. On the other hand, gaps, missed pathways, included metabolism of terpenoids and polyketides and lipid metabolism. The multi-omics resource generated in this work will set the basis for ongoing and future studies, bringing the Holm oak closer to model species, to obtain a better understanding of the molecular mechanisms underlying phenotypes of interest (productive, tolerant to environmental cues, nutraceutical value) and to select elite genotypes to be used in restoration and reforestation programs, especially in a future climate change scenario.

In this century we have assisted at an unimaginable expansion of proteomics, with continuous innovations and optimizations in methods, techniques, protocols, equipment, and associated bioinformatics tools. We have moved forward very fast from first (gel electrophoresis based), to second (based on isotopic or isobaric labelling), to third (shotgun or gel-free, label-free), and to fourth (targeted, mass-western, or SRM/MRM) generation techniques. This evolution is clearly observed in the literature since 1994, when the term "proteome" was first coined, with plant proteomics progressing at a much lower speed than human and other model organisms. The question behind this review is: Is gel electrophoresis an obsolete technique? Is it still alive? The answer is that gel electrophoresis is still a valid technique, with its own particularities, strengths, and weaknesses, "irreplaceable" in top-down experiments directed at investigating protein species, loci and allelic variants, and isoforms, as well as in the post-translational modifications and interactions studies; it is an excellent complementary and alternative approach that could lead us to achieve a deeper visualization and knowledge of the cell proteome. The past, present, and future of this technique is being reviewed. It is not pretended to discuss in detail technical aspects, referring to key original papers or previous reviews, but instead, how it has contributed, from a historical perspective, to plant proteomics and biology research. It is our personal congratulations to "Journal of Proteomics" that celebrates this year its 10th anniversary, and, at the same time, a tribute to those scientists who have contributed to the establishment and development of the gel electrophoresis technique and its application to proteomics and plant biology research. Their direct or indirect teaching has been very valuable to those of us who once decided to enter proteomics, with no access to any sophisticated and expensive equipment. This gel electrophoresis-based plant proteomics review is divided into the following sections: introduction, history, methodology, contribution to plant biology research, and future directions.

The Spanish dehesas have been severely affected by human activities that date to the prehistoric period and have suffered accelerated decline since the 1980s. Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) is a key component of this system, and its acorns provide an important food source for wildlife and domesticated livestock. Our earlier work showed structured variation in acorn morphology and biochemistry. Here, we used chloroplast and nuclear microsatellites to detect genetic structure among populations of Q. ilex from the major biogeographic regions of Andalusia. We found high levels of spatial differentiation with chloroplast DNA indicating little seed dispersal among populations. Spatial differentiation was weaker for nuclear DNA, presumably as a result of more widespread pollen dispersal and its larger effective population size. The Baetic Cordillera (C&aacute, diz) population consistently appeared well separated from populations of the northern Sierra Morena, suggesting that the Guadalquivir Valley has played an important role in determining population divergence. This may be, in part, evolutionary, as suggested by chloroplast DNA, and, in part, a result of human-induced population isolation, as Q. ilex has been removed from the Guadalquivir Valley. Evolutionary gene flow rates were greater than contemporary rates, which were limited to unidirectional gene flow from C&oacute, rdoba to other populations in the Sierra Morena and, surprisingly, to the southern population at Almer&iacute, a. The inconsistency between evolutionary and recent migration rates suggests an effect of anthropogenic activity over the last few generations of Q. ilex.

Proteomics, either considered as being a scientific discipline or a methodological approach, will be presented from a historical perspective, as the heir of the classical protein chemistry or biochemistry. In the last 20 years, classical techniques for protein extraction and resolution such as electrophoresis and liquid chromatography have been continuously improved and implemented with those of mass spectrometry, gene sequencing, and bioinformatics tools, providing a new horizon in molecular biology research, and revealing the never-before imagined complexity of the protein world. The application of proteomics to plant biology research will be briefly discussed, focusing on descriptive and comparative studies and on two-dimensional electrophoresis-based platforms. There will be sections devoted to the different steps in a standard proteomics workflow: protein extraction, fractionation, electrophoresis, mass spectrometry, and data analysis. As each technique and protocol has to be adapted and optimized to the experimental system under investigation, rather than providing specific detailed protocols, general guidelines are presented, with reference to previous original publications and reviews from the author’s group and the current relevant literature. More than to scientists with real expertise in proteomics, this chapter is dedicated to plant physiologists, who aim to enter the field.