Elevated CO2 alters photosynthesis, growth and susceptibility to powdery mildew of oak seedlings.

Elevated CO2 (eCO2) is a determinant factor of climate change and is known to alter plant processes such as physiology, growth and resistance to pathogens. Quercus robur, a tree species integrated in most forest regeneration strategies, shows high vulnerability to powdery mildew (PM) disease at the seedling stage. PM is present in most oak forests and it is considered a bottleneck for oak woodland regeneration. Our study aims to decipher the effect of eCO2 on plant responses to PM. Oak seedlings were grown in controlled environment at ambient (aCO2, ∼400 ppm) and eCO2 (∼1000 ppm), and infected with Erysiphe alphitoides, the causal agent of oak PM. Plant growth, physiological parameters and disease progression were monitored. In addition, to evaluate the effect of eCO2 on induced resistance (IR), these parameters were assessed after treatments with IR elicitor ß-aminobutyric acid (BABA). Our results show that eCO2 increases photosynthetic rates and aerial growth but in contrast, reduces root length. Importantly, under eCO2 seedlings were more susceptible to PM. Treatments with BABA protected seedlings against PM and this protection was maintained under eCO2. Moreover, irrespectively of the concentration of CO2, BABA did not significantly change aerial growth but resulted in longer radicular systems, thus mitigating the effect of eCO2 in root shortening. Our results demonstrate the impact of eCO2 in plant physiology, growth and defence, and warrant further biomolecular studies to unravel the mechanisms by which eCO2 increases oak seedling susceptibility to PM.

Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion.

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.

Scaling-up to understand tree-pathogen interactions: A steep, tough climb or a walk in the park?

Plants have proficient tools that allow them to survive interactions with pathogens. Upon attack, they respond with specific countermeasures, which are controlled by the immune system. However, defences can fail and this failure exposes plants to fast-spreading devastation. Trees face similar challenges to other plants and their immune system allows them to mount defences against pathogens. However, their slow growth, longevity, woodiness, and size can make trees a challenging system to study. Here, we review scientific successes in plant systems, highlight the key challenges and describe the enormous opportunities for pathology research in trees. We discuss the benefits that scaling-up our understanding on tree-pathogen interactions can provide in the fight against plant pathogenic threats.

Specific Protein Database Creation from Transcriptomics Data in Nonmodel Species: Holm Oak (Quercus ilex L.).

Proteomics encompasses efforts to identify all the proteins of a proteome, with most of studies about plant proteomics based on a bottom-up mass spectrometry (MS) strategy, in which the proteins are subjected to digestion by trypsin and the tryptic fragments are subjected to MS analysis. The identification of proteins from MS/MS spectra has been performed using different algorithms (Mascot, Sequest) against plant protein sequence databases such as UniProtKB or NCBI_Viridiplantae. But these databases are not the best choice for nonmodel species where they are underrepresented, resulting in poor identification rates. A high identification rate requires a sequenced and well-annotated genome of the species under investigation. For nonmodel organisms, the identification of proteins is challenging since, in the best of the cases, only hits or orthologs instead of gene products are identified. However, in the absence of a sequenced genome, this situation can be improved using transcriptome data to generate a specific species database to compare proteins. In this chapter, we report the protein database construction from RNA-Seq data in a nonmodel species, in this particular case Holm oak (Q. ilex).

Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Species: The Case of Holm Oak (Quercus ilex).

The proteomics of orphan, unsequenced, and recalcitrant organisms is highly challenging. This is the case of the typical Mediterranean forest tree Holm oak (Quercus ilex). Proteomics has moved on quite fast from the classical 2DE-MS to shotgun or gel-free/label-free approaches, with the latter possessing a series of advantages over the gel-based ones. Before translating proteomics data into biological knowledge, a few questions as to the analysis technique itself have to be answered including its confidence in protein identification and quantification. It is important to clearly differentiate a hit from an ortholog and gene product identification, with the difference depending on the database and the confidence parameters (score, number of peptides, and coverage). With respect to quantification and for comparative purposes it is important to make sure that we are within the linear dynamic range. For that, a calibration curve based on mass spectrometry analysis of a serial dilution of the extracts should be performed. Thus, just by validating our data with the aim of improving the quality of the analysis enables us to give a correct interpretation of our results. We show a method that aims to improve the confidence in protein identification and quantification in the orphan species Q. ilex using a shotgun proteomics approach.

Proteomic analysis of goat milk kefir: Profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity.

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.

Proteomics, Holm Oak (Quercus ilex L.) and Other Recalcitrant and Orphan Forest Tree Species: How do They See Each Other?

Proteomics has had a big impact on plant biology, considered as a valuable tool for several forest species, such as Quercus, Pines, Poplars, and Eucalyptus. This review assesses the potential and limitations of the proteomics approaches and is focused on Quercus ilex as a model species and other forest tree species. Proteomics has been used with Q. ilex since 2003 with the main aim of examining natural variability, developmental processes, and responses to biotic and abiotic stresses as in other species of the genus Quercus or Pinus. As with the progress in techniques in proteomics in other plant species, the research in Q. ilex moved from 2-DE based strategy to the latest gel-free shotgun workflows. Experimental design, protein extraction, mass spectrometric analysis, confidence levels of qualitative and quantitative proteomics data, and their interpretation are a true challenge with relation to forest tree species due to their extreme orphan and recalcitrant (non-orthodox) nature. Implementing a systems biology approach, it is time to validate proteomics data using complementary techniques and integrate it with the -omics and classical approaches. The full potential of the protein field in plant research is quite far from being entirely exploited. However, despite the methodological limitations present in proteomics, there is no doubt that this discipline has contributed to deeper knowledge of plant biology and, currently, is increasingly employed for translational purposes.

Gel electrophoresis-based plant proteomics: Past, present, and future. Happy 10th anniversary Journal of Proteomics!

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.

Holm oak proteomic response to water limitation at seedling establishment stage reveals specific changes in different plant parts as well as interaction between roots and cotyledons.

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.

A Multi-Omics Analysis Pipeline for the Metabolic Pathway Reconstruction in the Orphan Species Quercus ilex.

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.

Effects of olive root warming on potassium transport and plant growth.

Young olive (Olea europaea L.) plants generated from seed were grown in liquid hydroponic medium exposing the roots system for 33days or 24h to high temperature (37°C) while the aerial part to 25°C aiming to determine the prolonged and immediate effects of root warming on K+(Rb+) transport in the root and consequently on plant growth. The exposition of the root system to 37°C for 24h inhibited K+ (Rb+) transport from root to shoot having no effect on its uptake. However, when the root system was exposed permanently to 37°C both the K+ (Rb+) uptake and translocation to the aerial part were inhibited as well as the growth in all plants organs. The ability of the root system to recover K+ (Rb+) uptake and transport capacity after being exposed to high temperature was also evaluated. Plants grown in a root medium at 37°C for 31days were transferred to another at 25°C for 48 or 96h. The recovery of K+ (Rb+) root transport capacity after high root temperature was slow. Any signal of recovery was observed after 48h without stress: both potassium root uptake and subsequent transport to above organs were inhibited yet. Whereas 96h without stress led to restore potassium upward transport capacity although the uptake was partially inhibited yet. The results obtained in this study have shown that the root system of young olive plants is very sensitive to high temperature related to root potassium transport and growth of the plant. Taking into account the two processes involved in root potassium transport, the discharge of K+ to the xylem vessels was more affected than the uptake at the initial phase of high root temperature stress. However, it was the first process to be re-established during recovery. All this could explain the symptoms frequently observed in olive orchards when dry and high temperature spells occur: a reduction in shoots growth and leaves with low levels of potassium contents and dehydration symptoms.

Effect of moderate high temperature on the vegetative growth and potassium allocation in olive plants.

There is little information about the prolonged effect of a moderately high temperature on the growth of olive (Olea europaea L.). It has been suggested that when the temperature of the air rises above 35°C the shoot growth of olive is inhibited while there is any reference on how growth is affected when the soil is warmed. In order to examine these effects, mist-cuttings and young plants generated from seeds were grown under moderate high temperature (37°C) for 64 and 42days respectively. In our study, plant dry matter accumulation was reduced when the temperature of both the air and the root medium was moderately high. However, when the temperature of the root medium was 25°C, the inhibitory effect of air high temperature on plant growth was not observed. The exposure of both the aerial part and the root to moderate high temperature also reduced the accumulation of K+ in the stem and the root, the water use efficiency and leaf relative water content. However, when only the aerial part was exposed to moderate high temperature, the accumulation of K+ in the stem, the water use efficiency and leaf relative water content were not modified. The results from this study suggest that the olive is very efficient in regulating the water and potassium transport through the plant when only the atmosphere surrounding the aerial part is warmed up. However, an increase in the soil temperature decrease root K+ uptake and its transport to the aerial parts resulting in a reduction in shoot water status and growth.

Proteomics for exploiting diversity of lupin seed storage proteins and their use as nutraceuticals for health and welfare.

UNLABELLED: Lupins have a variety of both traditional and modern uses. In the last decade, reports assessing the benefits of lupin seed proteins have proliferated and, nowadays, the pharmaceutical industry is interested in lupin proteins for human health. Modern genomics and proteomics have hugely contributed to describing the diversity of lupin storage genes and, above all, proteins. Most of these studies have been centered on few edible lupin species. However, Lupinus genus comprises hundreds of species spread throughout the Old and New Worlds, and these resources have been scarcely explored and exploited. We present here a detailed review of the literature on the potential of lupin seed proteins as nutraceuticals, and the use of -omic tools to analyze seed storage polypeptides in main edible lupins and their diversity at the Lupinus inter- and intra-species level. In this sense, proteomics, more than any other, has been a key approach. Proteomics has shown that lupin seed protein diversity, where post-translational modifications yield a large number of peptide variants with a potential concern in bioactivity, goes far beyond gene diversity. The future extended use of second and third generation proteomics should definitely help to go deeper into coverage and characterization of lupin seed proteome. BIOLOGICAL SIGNIFICANCE: Some important topics concerning storage proteins from lupin seeds are presented and analyzed in an integrated way in this review. Proteomic approaches have been essential in characterizing lupin seed protein diversity, which goes far beyond gene diversity since the protein level adds to the latter differential proteolytic cleavage of conglutin pro-proteins and a diverse array of glycosylation forms and sites. Proteomics has also proved helpful for screening and studying Lupinus germplasm with the future aim of exploiting and improving food production, quality, and nutritional values.

A year (2014-2015) of plants in Proteomics journal. Progress in wet and dry methodologies, moving from protein catalogs, and the view of classic plant biochemists.

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.

Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes.

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage.

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

Fourteen years of plant proteomics reflected in Proteomics: moving from model species and 2DE-based approaches to orphan species and gel-free platforms.

In this article, the topic of plant proteomics is reviewed based on related papers published in the journal Proteomics since publication of the first issue in 2001. In total, around 300 original papers and 41 reviews published in Proteomics between 2000 and 2014 have been surveyed. Our main objective for this review is to help bridge the gap between plant biologists and proteomics technologists, two often very separate groups. Over the past years a number of reviews on plant proteomics have been published . To avoid repetition we have focused on more recent literature published after 2010, and have chosen to rather make continuous reference to older publications. The use of the latest proteomics techniques and their integration with other approaches in the "systems biology" direction are discussed more in detail. Finally we comment on the recent history, state of the art, and future directions of plant proteomics, using publications in Proteomics to illustrate the progress in the field. The review is organized into two major blocks, the first devoted to provide an overview of experimental systems (plants, plant organs, biological processes) and the second one to the methodology.