Analyzing differences in tolerance to drought in Quercus spp., and the characterization of these responses at the species and individual population level, are imperative for the selection of resilient elite genotypes in reforestation programs. The main objective of this work was to evaluate differences in the response and tolerance to water shortage under in five Quercus spp. and five Andalusian Q. ilex populations at the inter- and intraspecies level. Six-month-old seedlings grown in perlite were subjected to drought treatments by withholding water for 28 days under mean 37 °C temperature, 28 W m−2 solar irradiance, and 41% humidity. The use of perlite as the substrate enabled the establishment of severe drought stress with reduction in water availability from 73% (field capacity) to 28% (dryness), corresponding to matric potentials of 0 and −30 kPa. Damage symptoms, mortality rate, leaf water content, photosynthetic, and biochemical parameters (amino acids, sugars, phenolics, and pigments) were determined. At the phenotypic level, based on damage symptoms and mortality, Q. ilex behaved as the most drought tolerant species. Drought caused a significant decrease in leaf fluorescence, photosynthesis rate, and stomatal conductance in all Quercus spp. analyzed, being less pronounced in Q. ilex. There were not differences between irrigated and non-irrigated Q. ilex seedlings in the content of sugar and photosynthetic pigments, while the total amino acid and phenolic content significantly increased under drought conditions. As a response to drought, living Q. ilex seedlings adjust stomata opening and gas exchange, and keep hydrated, photosynthetically active, and metabolically competent. At the population level, based on damage symptoms, mortality, and physiological parameters, the eastern Andalusian populations were more tolerant than the western ones. These observations inform the basis for the selection of resilient genotypes to be used in breeding and reforestation programs.

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].

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.

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