Metabolic signature of nutrient conditions
The metabolic impact of nutrient conditions was analyzed using GC-MS profiling. 178 (leaves) and 187 (roots) analytes were identified and quantified. The multivariate statistical analysis by OPLS allowed facile discrimination of samples in both leaves (Fig. 4a) and roots (Fig. 5a). The multivariate statistical model was good and robust as shown by high R² and Q² values (0.94 and 0.87, respectively, in both leaves and roots), and highly significant (P CV-ANOVA<10-30 (K), < 10-22 (Ca) and <10-11(putrescine)). Axis 1 was driven by K conditions, while Ca and putrescine conditions were driven by both axes 2 and 3, showing some interaction in their metabolic effect. A specific analysis of putrescine content is provided in Fig. S2. When all samples were pooled together (old and new leaves), putrescine addition was not found to cause an important increase in tissue putrescine pool in leaves (on average, × 2) and roots (on average, × 1.25). In roots, putrescine was 2-fold lower under low Ca.
In leaves, low K conditions were associated with many highly significant changes, typically sugar (arabinose, ribose), putrescine, citrate and ascorbate accumulation and a decrease in glucose 6-phosphate, inorganic phosphate (Pi), threonate, amino acids (glutamate, aspartate) and their derivatives (cystathionine, β-alanine) (Fig. 4b). Ca conditions were not associated with many significant metabolites and only one metabolite increased by high Ca was above the Bonferroni threshold, tartarate (reduced form of oxaloacetate) (Fig. 4c). Low Ca was associated with an accumulation of organic acids of the tricarboxylic acid pathway (TCAP), such as citrate, isocitrate, fumarate and aconitate. Under low Ca, there was also more Pi and glutamine, suggesting a beneficial effect of low Ca on N and P nutrition. Very few metabolites were affected above the Bonferroni threshold by putrescine addition (Fig. 4d): putrescine homoserine and serine (increased) and cellobiose and gulonate (decreased). Interestingly, with a P -value lower than 0.001, tartarate appeared to be decreased by putrescine (thus counteracting the effect of high Ca), while sucrose increased.
Like in leaves, many metabolites were significantly affected by low K conditions in roots, including sugars (maltose, xylose, allose, gentiobiose), putrescine and ascorbate (increased), and aspartate, threonate, cystathionine, β-alanine (decreased) (Fig. 5b). Also, low Ca led to an accumulation in organic acids (glutarate, isocitrate, succinate, aconitate, citrate, citramalate) (Fig. 5c). Interestingly, spermidine (derivative of putrescine) was found to increase at high Ca. Very few metabolites were affected by putrescine addition: putrescine, glutamine, asparagine and methylaspartate (increased), and glutamine, fumarate, xylulose, glucuronate (decreased) (Fig. 5d).
Despite the similarities in the response to K, Ca or putrescine, metabolites associated with a significant K x putrescine effect were different in leaves and roots. Typically, under low K in leaves, putrescine addition led to a decrease in several sugars, polyols and shikimate, and an increase in glycolate (and, of course, putrescine) (Fig. 4e). In roots, putrescine addition decreased isoprenoids (phytol, tocopherol), sugars and sugar derivatives (2-oxogluconate, rhamnose, xylulose) (Fig. 5e).