Germination and Early Seedling Development in Quercus ilex Recalcitrant and Non-dormant Seeds: Targeted Transcriptional, Hormonal, and Sugar Analysis.

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0-S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1-S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds.

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.

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.

Improving the quality of protein identification in non-model species. Characterization of Quercus ilex seed and Pinus radiata needle proteomes by using SEQUEST and custom databases.

Nowadays the most used pipeline for protein identification consists in the comparison of the MS/MS spectra to reference databases. Search algorithms compare obtained spectra to an in silico digestion of a sequence database to find exact matches. In this context, the database has a paramount importance and will determine in a great deal the number of identifications and its quality, being this especially relevant for non-model plant species. Using a single Viridiplantae database (NCBI, UniProt) and TAIR is not the best choice for non-model species since they are underrepresented in databases resulting in poor identification rates. We demonstrate how it is possible to improve the rate and quality of identifications in two orphan species, Quercus ilex and Pinus radiata, by using SEQUEST and a combination of public (Viridiplantae NCBI, UniProt) and a custom-built specific database which contained 593,294 and 455,096 peptide sequences (Quercus and Pinus, respectively). These databases were built after gathering and processing (trimming, contiging, 6-frame translation) publicly available RNA sequences, mostly ESTs and NGS reads. A total of 149 and 1533 proteins were identified from Quercus seeds and Pinus needles, representing a 3.1- or 1.5-fold increase in the number of protein identifications and scores compared to the use of a single database. Since this approach greatly improves the identification rate, and is not significantly more complicated or time consuming than other approaches, we recommend its routine use when working with non-model species. BIOLOGICAL SIGNIFICANCE: In this work we demonstrate how the construction of a custom database (DB) gathering all available RNA sequences and its use in combination with Viridiplantae public DBs (NCBI, UniProt) significantly improve protein identification when working with non-model species. Protein identification rate and quality is higher to those obtained in routine procedures based on using only one database (commonly Viridiplantae from NCBI), as we demonstrated analyzing Quercus seeds and Pine needles. The proposed approach based on the building of a custom database is not difficult or time consuming, so we recommend its routine use when working with non-model species. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Standardization of data processing and statistical analysis in comparative plant proteomics experiment.

Two-dimensional gel electrophoresis remains the most widely used technique for protein separation in plant proteomics experiments. Despite the continuous technical advances and improvements in current 2-DE protocols, an adequate and correct experimental design and statistical analysis of the data tend to be ignored or not properly documented in current literature. Both proper experimental design and appropriate statistical analysis are requested in order to confidently discuss our results and to conclude from experimental data.In this chapter, we describe a model procedure for a correct experimental design and a complete statistical analysis of proteomic dataset. Our model procedure covers all of the steps in data mining and processing, starting with the data preprocessing (transformation, missing value imputation, definition of outliers) and univariate statistics (parametric and nonparametric tests), and finishing with multivariate statistics (clustering, heat-mapping, PCA, ICA, PLS-DA).

Back to Osborne. Sequential protein extraction and LC-MS analysis for the characterization of the Holm oak seed proteome.

It is impossible to capture in just one experiment all or most of the total set of protein species that constitute the cell's proteome. Thus, according to our results, and even considering that they depend on the experimental system carried out (plant, yeast, fungi, or bacteria), the best protein extraction protocol yielded less than 20 % of the total amount of proteins, as determined by the Kjeldahl method. For this reason, protein cataloguing and the whole proteome characterization require the use of firstly, fractionation techniques at the cellular, subcellular, protein, or peptide level, and secondly, the use of complementary approaches.Within our current research on Holm oak (Quercus ilex subsp. ballota), we aim to characterize its seed proteome. For that we have optimized an experimental workflow in which the Osborne sequential protein extraction (Osborne, Science 28:417-427, 1908) is combined with downstream electrophoretic protein separation or shotgun MS analysis. In general, it can be used to study any plant seed, as well as to investigate on seed maturation and germination, genotype characterization, allergens identification, food traceability, and substantial equivalence, among others.

Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi.

Phytophthora cinnamomi is one of the agents that trigger the decline syndrome in Quercus spp., this being a serious threat to Mediterranean Holm oak forest sustainability and reforestation programs. Quercus ilex responses to Phytophthora cinnamomi have been studied in one-year olds seedlings from two Andalucía provenances, assessing the physiological water status and photosynthesis-related parameters. Upon inoculation with mycelium a reduction in water content, chlorophyll fluorescence, stomatal conductance and gas exchange was observed along a 90 days post inoculation period in both provenances. The reduction was higher in the most susceptible (SSA) provenance, than in the most tolerant (PCO), being these typical plant responses to drought stress. Leaf protein profiles were analyzed in non-inoculated and inoculated seedlings from the two provenances by using a 2-DE coupled to MS proteomics strategy. Ninety seven proteins changing in abundance in response to the inoculation were successfully identified after MALDI-TOF-TOF analyses. The largest group of variable identified proteins were chloroplasts ones, and they were involved in the photosynthesis, Calvin cycle and carbohydrate metabolism. It was noted that a general tendency was a decrease in the protein abundance as a consequence of the inoculation, being it less accused in the least susceptible, the Northern provenance (PCO), than in the most susceptible, the Southern provenance (SSA). This trend is clearly manifested in photosynthesis, amino acid metabolism and stress/defence proteins. On the contrary, some proteins related to starch biosynthesis, glycolysis and stress related peroxiredoxin showed an increase upon inoculation. These changes in protein abundance were correlated to the estimated physiological parameters and have been frequently observed in plants subjected to drought stress.