Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

CRIBI Biotechnology Center University of Padova viale G Colombo 3 35131 Padua Italy

DAFNAE University of Padova Viale Università 16 AGRIPOLIS 1 35020 Legnaro PD Italy

Department of Biomedical Sciences University of Padova Via U Bassi 58 B 35131 Padua Italy

Department of Plant Biophysics and Biochemistry Biology Centre Institute of Plant Molecular Biology Czech Academy of Sciences Branišovská 31 1160 37005 České Budějovice Czech Republic

Institute for Multidisciplinary Research Department of Life Science University of Belgrade Kneza Višeslava 1 Belgrade 11030 Serbia

Institute of Molecular Genetics and Genetic Engineering Laboratory for Plant Molecular Biology University of Belgrade Vojvode Stepe 444a Belgrade 11042 Serbia

Proteomics Center University of Padova and Azienda Ospedaliera di Padova Via G Orus 2 B 35129 Padua Italy

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