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.