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

Palak Chaturvedi,María-Dolores Rey,Jesús V Jorrín-Novo,Victor M Guerrero-Sánchez,Bonoso San-Eufrasio,Ezequiel Darío Bigatton,María Ángeles Castillejo

doi:10.3390/ijms22063191

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.

Highlights

Holm oak (Quercus ilex) is the dominant tree species in natural forest ecosystems over the Western Mediterranean Basin, as well as in the agrosilvopastoral Spanish “dehesa”, which is environmentally, economically and socially important [1,2]
Shotgun analysis allowed a total of 4470 proteins to be identified in the Q. ilex leaf proteome (Supplementary Table S1; data are available via ProteomeXchange with identifier PXD023782) of which 2920 fulfilled the following criterion for confident identification: XCorr ≥ 2 and at least two different peptides per protein
The proposed targeted strategy is aimed at identifying peptides associated with the response of Q. ilex to drought stress

Summary

Introduction

Holm oak (Quercus ilex) is the dominant tree species in natural forest ecosystems over the Western Mediterranean Basin, as well as in the agrosilvopastoral Spanish “dehesa”, which is environmentally, economically and socially important [1,2] This species is highly adaptable to drought and to the high temperatures and irradiation typical of Southern Spain. The response of plants to stress-related situations may, in theory, be improved by characterizing their biodiversity and selecting elite genotypes based on specific molecular markers This approach can be quite challenging with orphan species, such as holm oak, which has a still incompletely sequenced genome and largely unexplored molecular features [11,12,13,14,15,16,17,18,19,20,21]. Species-specific improved databases, such as the recently compiled holm oak transcriptome database [16,17], and other sequenced Quercus species databases, such as those for Q. robur [25] and Q. suber [26], have substantially expanded available knowledge of holm oak biology

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