Protein Extraction and 2D Electrophoresis

Proteins were extracted using the method described by Hurkman and Tanaka (1986) (link) with certain modifications. The sample preparation procedure as well as 2D electrophoresis conditions used for separation are shown in Supplementary Table S1, and were also described in details previously (Chmielewska et al., 2016 (link)). Plant material was extracted from two biological replications and two technical repetitions were prepared from each biological sample. Image Master 2D Platinum 7.0 software (IMP7, GE Healthcare, USA) was used for quantitative gel analysis. Protein spots’ relative volume (%vol.) was quantified. This parameter is relatively independent of sample loading and staining variation. In the case of roots, we could not proceed with 11 RILs due to insufficient material. For each genotype and tissue, four gels in control and four in drought conditions were analysed. The images of 2D electrophoretic gels were deposited in the Dryad Digital Repository (https://doi.org/10.5061/dryad.6st3v7r; Rodziewicz et al., 2019 (link)).