Figure 4

Classification of the different samples according to multivariate methods. (A) Principal componet analysis (PCA) of the integrated proteomic, metabolomic, and physiological datasets. Glutamate family enzymes (ASS, ASL, OCT, NAG), ammonia metabolism (Cre13.g592200.t1.2), purine biosynthesis proteins (Cre07.g318750.t1.2, Cre08.g364800.t1.2), NADH:ubiquinone oxidoreductase, cGMP-dependent kinases (Cre03.g199050.t1.2), glycolysis enzymes (PK, GAP-DH, PEPC), glyceraldehyde-3P-DH, glycerol, and C18:3 showed high correlations to PC1. Calvin cycle proteins (SBPase, PPE), chloroplastic ATPase, amino acid degradation, polyamine synthase, fatty acid elongation, catalases, and aspartic acid showed a negative correlation to PC1. Fresh weight, alanine, beta oxidation-related proteins (Acyl-CoA oxidases, HADH), oxidoreductases (Cre16.g677950.t1.3, g13806.t1, g4488.t1, g9426.t1), signal peptide and protein peptidases, and tetrapyrrole biosynthesis proteins showed a high correlation to PC2, while organic acids (fumaric and glyceric), phosphate, and photosynthesis-related enzymes (light reaction and carbon fixation), showed a negative correlation. These variables were used to infer the biological meaning of the principal components 1 and 2. Loading matrix is available in Additional file 2: Table S4. (B) Hierarchical clustering and heatmap of the analyzed proteins grouped by functional category according to MapMan. Three different clusters (0, 5 h, and 96 h, 24 h, and 72 and 77 h) can be distinguished, showing the different degrees of response to N starvation. The aggrupation of the 96-h and 5-h samples remarks the effect of the N repletion over the cultures. (C) sPLS-based network, showing the significant interaction between proteins and metabolites. Three major clusters can be distinguished, corresponding to fresh weight, glycerol/C18:2/C16, and N metabolism, which are mixed in the image. Fv/Fm and C16 are outside these groups acting as a link. For further details see Results and Discussion. This complex network is depicted in Additional file 6: Figure S5D, Additional file 10: Figures S9 and Additional file 11: Figure S10.