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

Abstract

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.

Table_1.xlsx

“…Thus, the two populations differed in the number of proteins but not in the functional categories. The largest groups of confidence and variable upaccumulated proteins in the two populations were those of amino acid metabolism; carbohydrate metabolism; folding, sorting, and degradation; ROS scavenging; and secondary metabolism (Sghaier-Hammami et al, 2013;Valero-Galván et al, 2013;Hildebrandt, 2018;San-Eufrasio et al, 2021). In contrast, the largest number of downaccumulated proteins in both populations, which contributed more to the variability, was that of the photosynthesis (Kapoor et al, 2020), which is in line with the above-described results for physiological parameters.…”

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

“…Thus, the two populations differed in the number of proteins but not in the functional categories. The largest groups of confidence and variable upaccumulated proteins in the two populations were those of amino acid metabolism; carbohydrate metabolism; folding, sorting, and degradation; ROS scavenging; and secondary metabolism (Sghaier-Hammami et al, 2013 ; Valero-Galván et al, 2013 ; Hildebrandt, 2018 ; San-Eufrasio et al, 2021 ). In contrast, the largest number of downaccumulated proteins in both populations, which contributed more to the variability, was that of the photosynthesis (Kapoor et al, 2020 ), which is in line with the above-described results for physiological parameters.…”

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

“…The only resulting band was excised and digested with trypsin and the peptides thus obtained were dissolved in 50 μl of a 4% acetonitrile/0.25% formic acid mixture for loading into an Ultimate 3,000 nano-LC-MS-UHPLC- Orbitrap Fusion instrument from Thermo Fisher Scientific (Waltham, MA, United States; Gómez-Gálvez et al, 2020 ). MS/MS data were processed with the software Proteome Discoverer v. 2.3, also from Thermo Fischer Scientific, and identified by using the SEQUEST algorithm against the species-specific database of Q. ilex developed from the transcriptome ( Guerrero-Sánchez et al, 2017 , 2019 , 2021 ), using the setting reported by San-Eufrasio et al (2021) . Proteins were quantified in relative form from the peak areas for the precursor ions, using the three strongest peptide ion signals for each protein ( Al Shweiki et al, 2017 ).…”