Proteomic analysis of plant material
Extraction of leaf proteins and analysis of protein content using gel-free LC-MS/MS was carried out as described by Miller et al.(2017). Briefly, frozen ground leaf samples (4 replicates per treatment) were extracted in a buffer containing Rapigest. After reduction, alkylation and trypsin digestion, samples were analysed by LC-MS/MS using an UltiMate® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, MA, USA). Raw data were analysed in Progenesis and peptide assignments were made using Mascot. A principal component analysis (PCA) was performed in the R software package using log2 scaled protein intensities. Proteins were considered to have significantly changed in abundance when a p value of <0.05 was reached, with a fold change of 1.2 or greater. For hierarchical clustering analysis, log2 scaled protein values were used. Hierarchical clustering was performed using Euclidean distance and the complete linkages method. For heatmap/cluster analysis, fold change data were calculated relative to the wild type Col-0 at LL and log2 scaled. A heatmap was then generated using the heatmap.2 package in R software, using the default settings.