Identification of N deficiency responsive root PM proteins
A total of 1512 unique peptides corresponding to 215 proteins were identified after subjecting the root PMe fraction to label-free LC-MS. The PCA of the LC-MS data shows that the first principal component was responsible for 75.3% of the explained variance and the second for the 14.4% (Supplementary Fig. S4). The clustering of the biological replicates in the scores plot demonstrates that the major variance in the dataset was due to the differences displayed between the contrasting cultivars under N deficiency, whereas their protein profiles were similar under N-sufficient conditions. About 33% of the overall identified proteins were predicted to harbor a transmembrane helix (Supplementary Fig. S5), of which 39 proteins harbored at least two transmembrane domains. N deficiency significantly induced changes in the abundance for 65 of the 215 identified proteins (Table 1).
K-means clustering assembled the differentially changed proteins into seven groups differing in their abundance patterns (Fig. 2). A single protein, namely defensin-like protein was assigned to cluster I, showing the lowest abundance in the sensitive cultivar ‘Lambada’ under N sufficiency. Cluster II comprised two proteins decreasing in abundance in ‘Lambada’ under N deficiency. Cluster III grouped two proteins displaying a decreased abundance under N deficiency in the tolerant cultivar ‘Topas’ and unchanged, low abundance in the sensitive cultivar ‘Lambada’. Cluster IV comprised 22 proteins, which showed an increased abundance under N deficiency in ‘Topas’, but not in ‘Lambada’. The overrepresented functional assignments in this cluster were ‘transporters’ (12 proteins) and ‘protein destination and storage’ (three proteins). Two proteins grouped in cluster V were more abundant in ‘Lambada’ under both N regimes. In cluster VI, eight proteins participating in various biological processes, showed a decreased abundance in the tolerant cultivar. Finally, cluster VII included 28 proteins which decreased in both cultivars under N deficiency. These included proteins assigned to the categories ‘transporters’ (five proteins), ‘signal transduction’ (three proteins), ‘cell structure’ (three proteins) and ‘energy’ (three proteins). The proteins that were exclusively increased in the tolerant genotype ‘Topas’ under N deficiency (Cluster IV), showing the highest contribution to the variance explained by the first principal component on the PCA (Supplementary Fig. S4) and harboring at least one transmembrane helix, were considered as integral root PM proteins likely involved in potato N deficiency tolerance.