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.

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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.

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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.

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.

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.