How temperature modulates the expression of pathogenesis-related molecules of the cross-kingdom pathogen Lasiodiplodia hormozganensis.

Lasiodiplodia hormozganensis, initially recognized as a fungal plant pathogen, is recognized now acknowledged as a potential threat to humans. However, our understanding of the pathogenesis mechanisms of Lasiodiplodia species remains limited, and the impact of temperature on its pathogenicity is unclear. This study aims to elucidate the effects of temperature on the biology of L. hormozganensis, focusing on the expression of pathogenesis-related molecules and its ability to function as a cross-kingdom pathogen. We conducted experiments at two different temperatures, 25 and 37 °C, analyzing the proteome and transcriptome of L. hormozganensis. Using strain CBS339.90, initially identified as L. theobromae but confirmed through ITS and tef1-α sequence analysis to be L. hormozganensis, we aimed to understand the fungus's protein expression under varying temperature conditions. Results from the functional analysis of the secretome at 25 °C showed a noteworthy presence of proteins related to carbohydrate metabolism, catabolism, plant cell wall degradation, and pathogenesis. However, when grown at 37 °C, the fungus exhibited an increased production of stress response and pathogenesis-related proteins. Our findings identified various pathways crucial for pathogenesis in both plants and humans, suggesting that L. hormozganensis possesses the genetic foundation to infect both hosts. Specific pathogenesis-related proteins, including the phytotoxin snodprot1, aspartic protease aspergillopepsin, and virulence protein SSD1, were also identified. Concluding, we propose a possible mechanism of how L. hormozganensis adapts to different temperatures. The shift in temperature results in the expression of genes that favor human related pathogenesis molecules.

Proteomic and Metabolomic Analysis of the Quercus ilex-Phytophthora cinnamomi Pathosystem Reveals a Population-Specific Response, Independent of Co-Occurrence of Drought.

Holm oak (Quercus ilex) is considered to be one of the major structural elements of Mediterranean forests and the agrosilvopastoral Spanish "dehesa", making it an outstanding example of ecological and socioeconomic sustainability in forest ecosystems. The exotic Phytophthora cinnamomi is one of the most aggressive pathogens of woody species and, together with drought, is considered to be one of the main drivers of holm oak decline. The effect of and response to P. cinnamomi inoculation were studied in the offspring of mother trees from two Andalusian populations, Cordoba and Huelva. At the two locations, acorns collected from both symptomatic (damaged) and asymptomatic (apparently healthy) trees were sampled. Damage symptoms, mortality, and chlorophyll fluorescence were evaluated in seedlings inoculated under humid and drought conditions. The effect and response depended on the population and were more apparent in Huelva than in Cordoba. An integrated proteomic and metabolomic analysis revealed the involvement of different metabolic pathways in response to the pathogen in both populations, including amino acid metabolism pathways in Huelva, and terpenoid and flavonoid biosynthesis in Cordoba. However, no differential response was observed between seedlings inoculated under humid and drought conditions. A protective mechanism of the photosynthetic apparatus was activated in response to defective photosynthetic activity in inoculated plants, which seemed to be more efficient in the Cordoba population. In addition, enzymes and metabolites of the phenylpropanoid and flavonoid biosynthesis pathways may have conferred higher resistance in the Cordoba population. Some enzymes are proposed as markers of resilience, among which glyoxalase I, glutathione reductase, thioredoxin reductase, and cinnamyl alcohol dehydrogenase are candidates.

Comparing and integrating TMT-SPS-MS3 and label-free quantitative approaches for proteomics scrutiny in recalcitrant Mango (Mangifera indica L.) peel tissue during postharvest period.

Despite substantial advances in the use of proteomic technologies, their widespread application in fruit tissues of non-model and recalcitrant species remains limited. This hampers the understanding of critical molecular events during the postharvest period of fleshy tropical fruits. Therefore, we evaluated label-free quantitation (LFQ) and TMT-SPS-MS3 (TMT) approaches to analyse changes in the protein profile of mango peels during postharvest period. We compared two extraction methods (phenol and chloroform/methanol) and two peptide fractionation schemes (SCX and HPRP). We accurately identified 3065 proteins, of which, 1492 were differentially accumulated over at 6 days after harvesting (DAH). Both LFQ and TMT approaches share 210 differential proteins including cell wall proteins associated with fruit softening, as well as aroma and flavour-related proteins, which were increased during postharvest period. The phenolic protein extraction and the high-pH reverse-phase peptide fractionation was the most effective pipeline for relative quantification. Nevertheless, the information provided by the other tested strategies was significantly complementary. Besides, LFQ spectra allowed us to track down intact N-glycopeptides corroborating N-glycosylations on the surface of a desiccation-related protein. This work represents the largest proteomic comparison of mango peels during postharvest period made so far, shedding light on the molecular foundation of edible fruit during ripening.

State-of-the-Art Molecular Plant Biology Research in Spain.

Molecular plant biology is the study of the molecular basis of plant life [...].

A first draft genome of holm oak (Quercus ilex subsp. ballota), the most representative species of the Mediterranean forest and the Spanish agrosylvopastoral ecosystem "dehesa".

The holm oak (Quercus ilex subsp. ballota) is the most representative species of the Mediterranean Basin and the agrosylvopastoral Spanish "dehesa" ecosystem. Being part of our life, culture, and subsistence since ancient times, it has significant environmental and economic importance. More recently, there has been a renewed interest in using the Q. ilex acorn 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 in the short and medium term. Breeding programs informed by the selection of elite genotypes seem to be the most plausible biotechnological solution to rescue populations under threat. To achieve this and other downstream analyses, we need a high-quality and well-annotated Q. ilex reference genome. Here, we introduce the first draft genome assembly of Q. ilex using long-read sequencing (PacBio). The assembled nuclear haploid genome had 530 contigs totaling 842.2 Mbp (N50 = 3.3 Mbp), of which 448.7 Mb (53%) were repetitive sequences. We annotated 39,443 protein-coding genes of which 94.80% were complete and single-copy genes. Phylogenetic analyses showed no evidence of a recent whole-genome duplication, and high synteny of the 12 chromosomes between Q. ilex and Quercus lobata and between Q. ilex and Quercus robur. 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 with resilience against oak decline syndrome and climate change and higher acorn productivity and nutraceutical value.

Proteomics research in forest trees: A 2012-2022 update.

This review is a compilation of proteomic studies on forest tree species published in the last decade (2012-2022), mostly focused on the most investigated species, including Eucalyptus, Pinus, and Quercus. Improvements in equipment, platforms, and methods in addition to the increasing availability of genomic data have favored the biological knowledge of these species at the molecular, organismal, and community levels. Integration of proteomics with physiological, biochemical and other large-scale omics in the direction of the Systems Biology, will provide a comprehensive understanding of different biological processes, from growth and development to responses to biotic and abiotic stresses. As main issue we envisage that proteomics in long-living plants will thrive light on the plant responses and resilience to global climate change, contributing to climate mitigation strategies and molecular breeding programs. Proteomics not only will provide a molecular knowledge of the mechanisms of resilience to either biotic or abiotic stresses, but also will allow the identification on key gene products and its interaction. Proteomics research has also a translational character being applied to the characterization of the variability and biodiversity, as well as to wood and non-wood derived products, traceability, allergen and bioactive peptides identification, among others. Even thought, the full potential of proteomics is far from being fully exploited in forest tree research, with PTMs and interactomics being reserved to plant model systems. The most outstanding achievements in forest tree proteomics in the last decade as well as prospects are discussed.

Multiomic Data Integration in the Analysis of Drought-Responsive Mechanisms in Quercus ilex Seedlings.

The integrated analysis of different omic layers can provide new knowledge not provided by their individual analysis. This approach is also necessary to validate data and reveal post-transcriptional and post-translational mechanisms of gene expression regulation. In this work, we validated the possibility of applying this approach to non-model species such as Quercus ilex. Transcriptomics, proteomics, and metabolomics from Q. ilex seedlings subjected to drought-like conditions under the typical summer conditions in southern Spain were integrated using a non-targeted approach. Two integrative approaches, PCA and DIABLO, were used and compared. Both approaches seek to reduce dimensionality, preserving the maximum information. DIABLO also allows one to infer interconnections between the different omic layers. For easy visualization and analysis, these interconnections were analyzed using functional and statistical networks. We were able to validate results obtained by analyzing the omic layers separately. We identified the importance of protein homeostasis with numerous protease and chaperones in the networks. We also discovered new key processes, such as transcriptional control, and identified the key function of transcription factors, such as DREB2A, WRKY65, and CONSTANS, in the early response to drought.

Multiomics Molecular Research into the Recalcitrant and Orphan Quercus ilex Tree Species: Why, What for, and How.

The holm oak (Quercus ilex L.) is the dominant tree species of the Mediterranean forest and the Spanish agrosilvopastoral ecosystem, "dehesa." It has been, since the prehistoric period, an important part of the Iberian population from a social, cultural, and religious point of view, providing an ample variety of goods and services, and forming the basis of the economy in rural areas. Currently, there is renewed interest in its use for dietary diversification and sustainable food production. It is part of cultural richness, both economically (tangible) and environmentally (intangible), and must be preserved for future generations. However, a worrisome degradation of the species and associated ecosystems is occurring, observed in an increase in tree decline and mortality, which requires urgent action. Breeding programs based on the selection of elite genotypes by molecular markers is the only plausible biotechnological approach. To this end, the authors' group started, in 2004, a research line aimed at characterizing the molecular biology of Q. ilex. It has been a challenging task due to its biological characteristics (long life cycle, allogamous, high phenotypic variability) and recalcitrant nature. The biology of this species has been characterized following the central dogma of molecular biology using the omics cascade. Molecular responses to biotic and abiotic stresses, as well as seed maturation and germination, are the two main objectives of our research. The contributions of the group to the knowledge of the species at the level of DNA-based markers, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are discussed here. Moreover, data are compared with those reported for Quercus spp. All omics data generated, and the genome of Q. ilex available, will be integrated with morphological and physiological data in the systems biology direction. Thus, we will propose possible molecular markers related to resilient and productive genotypes to be used in reforestation programs. In addition, possible markers related to the nutritional value of acorn and derivate products, as well as bioactive compounds (peptides and phenolics) and allergens, will be suggested. Subsequently, the selected molecular markers will be validated by both genome-wide association and functional genomic analyses.

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS, being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC-MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60-260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60-140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.

Effect and Response of Quercus ilex subsp. ballota [Desf.] Samp. Seedlings From Three Contrasting Andalusian Populations to Individual and Combined Phytophthora cinnamomi and Drought Stresses.

Quercus ilex L. is the dominant species in the Mediterranean forest and agrosilvopastoral ecosystem "dehesa." Currently, this forest species is threatened by natural and anthropogenic agents, especially by the decline syndrome, which is caused by Phytophthora cinnamomi and drought periods. Although the morphological and physiological responses of Q. ilex to combined stress (P. cinnamomi and drought) have been examined already, little is known at the molecular level. In this study, we studied the effect and response of 8-month seedlings from three contrasting Andalusian populations (Seville [Se], Granada [Gr], and Almeria [Al]) to the individual and combined stresses of P. cinnamomi and drought from morphological, physiological, biochemical, and proteomics data. Whereas, seedling damage (leaf chlorosis and necrosis) and mortality were greater under the combined stresses in the three populations, the effect of each individual stress was population-dependent. Resilient individuals were found in all the populations at different percentages. The decrease in leaf chlorophyll fluorescence, photosynthetic activity, and stomatal conductance observed in undamaged seedlings was greater in the presence of both stresses, the three populations responding similarly to drought and P. cinnamomi. Biochemical and proteomic analyses of undamaged seedlings from the two most markedly contrasting populations (Se and Al) revealed the absence of significant differences in the contents in photosynthetic pigments, amino acids, and phenolics among treatments. The Se and Al populations exhibited changes in protein profile in response to the different treatments, with 83 variable proteins in the former population and 223 in the latter. Variable proteins belonged to 16 different functional groups, the best represented among which were protein folding, sorting and degradation, carbohydrate, amino acid, and secondary metabolism, photosynthesis, and ROS scavenging. While photosynthetic proteins were mainly downaccumulated, those of stress-responsive were upaccumulated. Although no treatment-specific response was observed in any functional group, differences in abundance were especially marked under the combined stresses. The following variable proteins are proposed as putative markers for resilience in Q. ilex, namely, aldehyde dehydrogenase, glucose-6-phosphate isomerase, 50S ribosomal protein L5, and α-1,4-glucan-protein synthase [UDP-forming].

Why Consumers Prefer Green Friariello Pepper: Changes in the Protein and Metabolite Profiles Along the Ripening.

Fruit ripening is a physiologically complex process altering texture, color, flavor, nutritional value, and aroma. However, some fruits are consumed at an early stage of ripening due to the very peculiar characteristics varying during ripening. An example is a particular ecotype of pepper, the Friariello pepper, among the most important representatives of Campania (Southern Italy) agro-alimentary culture. In this study, for the first time, the physiological variations during Friariello ripening (green, veraison, and fully ripe) were evaluated by hyphenated mass spectrometric techniques in a proteomic and metabolomic approach. We found that Lutein and Thaumatin are particularly abundant in the green Friariello. Friariello at an early stage of ripening, is rich in volatile compounds like butanol, 1 3 5-cycloheptatriene, dimethylheptane, α-pinene, furan-2-penthyl, ethylhexanol, 3-carene, detected by gas chromatography-mass spectrometry (GC-MS) analysis, which give it the peculiar fresh and pleasant taste. The detected features of Friariello may justify its preferential consumption in the early ripening stage and outline new knowledge aimed at preserving specific agro-cultural heritage.

Interspecific Variation between the American Quercus virginiana and Mediterranean Quercus Species in Terms of Seed Nutritional Composition, Phytochemical Content, and Antioxidant Activity.

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.

Proteomics Data Analysis for the Identification of Proteins and Derived Proteotypic Peptides of Potential Use as Putative Drought Tolerance Markers for Quercus ilex.

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.

Combining P and Zn fertilization to enhance yield and grain quality in maize grown on Mediterranean soils.

The main aim of this study was to elucidate the effect of individual and joint fertilization with P and Zn on maize plants grown on typical Mediterranean soils with a limited Zn availability. For this purpose, we examined the effects of P and Zn fertilization individually and in combination on growth, yield and grain protein content in maize grown in pots filled with three different Mediterranean soils (LCV, FER and INM). Phosphorus and Zn translocation to grain was impaired, and aboveground dry matter and yield at harvest reduced by 8-85% (LCV and FER), in plants treated with Zn or P alone relative to unfertilized (control) plants. In contrast, joint fertilization with P and Zn enhanced translocation of these nutrients to grain and significantly increased aboveground dry matter (30% in LCV, 50% in FER and 250% in INM) and grain Zn availability in comparison with control plants. Also, joint application of both nutrients significantly increased grain P (LCV) and Zn (LCV and FER) use efficiency relative P and Zn, respectively, alone. Yield was increased between 31% in LCV and 121% in FER relative to control plants, albeit not significantly. Fertilization with P or Zn significantly influenced the abundance of specific proteins affecting grain quality (viz., storage, lys-rich and cell wall proteins), which were more abundant in mature grains from plants fertilized with Zn alone and, to a lesser extent, P + Zn. Sustainable strategies in agriculture should consider P-Zn interactions in maize grown on soils with a limited availability of Zn, where Zn fertilization is crucial to ensure grain quality.

Plant Proteomic Research 3.0: Challenges and Perspectives.

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

Phytochemical composition and variability in Quercus ilex acorn morphotypes as determined by NIRS and MS-based approaches.

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.

GeLC-Orbitrap/MS and 2-DE-MALDI-TOF/TOF comparative proteomics analysis of seed cotyledons from the non-orthodox Quercus ilex tree species.

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.

Application and optimization of label-free shotgun approaches in the study of Quercus ilex.

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.

Dissecting the Seed Maturation and Germination Processes in the Non-Orthodox Quercus ilex Species Based on Protein Signatures as Revealed by 2-DE Coupled to MALDI-TOF/TOF Proteomics Strategy.

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.