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.

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.