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.