Correlation based clustering among metabolites in leave and roots P. pinnata under salt stress
In order to explore the relationship among the metabolites in Pongamia subjected to salt stress, HAC (hierarchical cluster) analysis was performed on a total 60,492 elements of two different tissues leaf (30,246) and root (30,246) which include two different treatments 300 mM NaCl (15,123), 500 mM NaCl (15,123) and three time points 1, 4 and 8DAS (71 X 71 metabolite; 5041) (Figures 3 and 4). Correlation analysis was performed with Pearson correlation coefficients for each metabolite pair (Moing et al., 2011; Sánchez et al., 2012; Lombardo et al., 2011). The correlation values were clustered into six groups based on treatment time e.g. leaves of 300 mM NaCl treatment at 1DAS clustered into a group (Figure 3A and D; order of metabolite names and abbreviations were given in the Supplementary Table 3), leaves of 300 mM NaCl treatment at 4DAS clustered into a group (Figure 3B and E; Supplementary Table 3) and leaves of 300 mM NaCl treatment at 8DAS clustered into another group (Figure 3C and F; Supplementary Table 3). In order to simplify the graphics, only strong correlations (r≥0.6) were showed separately in the tables. Similarly, leaves of 500 mM NaCl at 1DAS clustered into one group (Figure 3G and J; order of metabolite names and abbreviations were given in the Supplementary Table 4), leaves of 500 mM NaCl at 4DAS clustered into one group (Figure 3H and K; Supplementary Table 4) and leaves of 500 mM NaCl at 8DAS clustered into one group (Figure 3I and L; Supplementary Table 4). In leaves of 300 mM NaCl treated plants, we observed high correlations within the organic acid group (e.g. CA:CAA:NA, 5HPip:Pip) and polyols (MI:MT). Although a significant correlation was not observed within the amino acid group, these levels showed strong interaction with organic acids (Asp:CA:NA:CAA, Asn:Pip:5HPip, GABA:MA) in leaves of 300 mM NaCl treated plants. Additionally, a strong positive correlation was observed between several metabolites such as fructose, myristate, mannitol and myo-inositol in leaves of 300 mM NaCl treated plants. In 500 mM NaCl treated plants, a significant correlation was observed within the amino acids group (Asp:Glu:Oxopro, Asn:Hse, Leu:Phe), organic acid group (e.g. 5HPip:CA:CAA, IBua:oC) and carbohydrate group (Xyl:pGlc, Man:pFru) at all-time points. Carbohydrates also showed significant interaction with other metabolites (Suc:Val, Xyl:SA:Pi, pGlc:SA) in 500 mM NaCl treated plants. Additionally, several metabolites showed a significant interaction within each other (e.g. Asp:Oxopro:Glu:5Hpip:CA:CAA:Grl-g, Man:pFru:MI:MT:oC:IBu, Asn:Hse:Pip) across all time points.
In the root dataset, the correlations among the metabolites were less pronounced when compared with leaf datasets. In roots of 300 mM NaCl treated plants, most of the metabolite levels showed significant correlation with each other (e.g. Glu:Oxopro:Thr:Phe:Hse:Ara:5Hpip:oC:Oxa:Pdo, PA:MytA:Gal:Gri:Met-SA:MT:Pi:Phytol:GlcNAc,pGlc:ThA:PI:pTag, Rib:Pyr:Glta:Eta, Suc:GA:Fru, HA:MI:SA) (Figure 4A-F; names and abbreviations were given in the Supplementary Table 5). In 500 mM NaCl treated plants, a strong positive correlation was observed within the organic acid group (SA:Oxa) and carbohydrate group (pGlc:Fru) across all the time points (Figure 4G-L; names and abbreviations were given in the Supplementary Table 6). In addition, several metabolites were associated significantly within each other (e.g. Oxopro:Asn:Thr:Phe:CAA:5Hpip:IBua:pFru:Thy, Ala:ThA, LA:Asu:PI, Rib:AA, HA:Pdo:Eta, Ba:Gal:Gri:PA, MytA:MT:Phytol) in 500 mM NaCl treated plants at all-time points.