Metabolic responses to P-stress
Ion, lipid and primary metabolite levels were determined to uncover metabolic responses of switchgrass to P limitation. Levels (mg/kg tissue) of all anions and cations tested changed significantly in shoots and/or roots in response to P-stress. Pi levels decreased substantially in both shoots (up to 3-fold) and roots (>10-fold) under P-stress, while nitrate and sulfate levels tended to increase in these organs and conditions (Figure 3a ). In contrast, chloride and malate levels tended to decrease under P-stress, possibly in response to the increasing levels of nitrate in order to maintain charge balance. In this context, potassium (K) and sodium (Na) levels decreased significantly in shoots and/or roots under P-stress.
P limitation affected lipid composition of switchgrass shoots and roots (Figure 3b ). Generally, phospholipid levels, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) decreased with increasing P-stress, in both shoots and roots, with the largest decreases observed under severe P-stress. In contrast, non-P lipids, including digalactosyl diacylglycerol (DGDG), monogalactosyl diacylglycerol (MGDG) and sulphoquinovosyl diacylglycerol (SQDG) increased significantly under moderate and severe P stress compared to P-replete roots. Interestingly, while these glyco- and sulfolipids increased under mild and moderate P-stress in shoots, their levels in severely P-stressed shoots were more similar to those in P-sufficient plants. Levels of triacylglycerol (TAG) remained stable under mild or moderate P-stress in shoots and declined by ~30% under severe P-stress, while levels of TAG increased in roots with increasing P-stress.
Primary metabolites were profiled by GC/MS to gain insight into the metabolic consequences of P-stress. A total of 95 compounds were identified, 55 of which were selected after t-test filter (P <0.05) for further principal component analysis (PCA;Figure 3c ). Principal component 1 (PC1) and PC2 accounted for 52 and 38 % of the total variation, respectively. PCA of the metabolite data revealed distinct metabolic signatures for shoots and roots, and at different levels of P-stress (Figure 3c ). Profiled metabolites can be classified into five categories, including phosphate, sugars and sugar alcohols, organic acids, amino acids, and miscellaneous metabolites. In general, most sugars and sugar alcohols decreased with increasing P stress, with a few exceptions including sucrose, gluconolactone, erythritol and erythrose. Organic acid levels increased under mild and moderate P-stress in both shoots and roots, although this trend reversed under severe P-stress, especially in shoots. Likewise, levels of most amino acids increased with mild and moderate P-stress, and declined especially in shoots under severe P-stress (Figure 4 ).