Additional file 1 of Metabolic alterations provide insights into Stylosanthes roots responding to phosphorus deficiency

Additional file 1: Figure S1 Growth performance of stylo at two P levels. Plants were grown in hydroponics for 15 d with (+Pi) or without (−Pi) 250 μmol/L KH2PO4 addition. Each bottle contained six plants. Figure S2 Effects of Pi availability on the increase ratios of root parameters. (a) Total root length. (b) Root surface area. (c) Root volume. After precultured under +Pi (250 μmol/L K2HPO4) for 7 d, stylo seedlings were transferred into nutrient solution with (+Pi) or without (−Pi) 250 μmol/L KH2PO4 additions for 7, 10, 15, 20 d. Each bar represents the mean of six replicates with SE (n = 6). Different lowercase letters indicate significant difference among groups (P < 0.05). Ratio of increase under -Pi (%) = [(−Pi - + Pi)/+Pi] * 100. Figure S3 Ratio of root/shoot in stylo at two P treatments. Uniform 7 d old stylo seedling was transferred into nutrient solution with (+Pi) or without (−Pi) 250 μmol/L KH2PO4 additions for 15 d. Asterisks represent significant differences between +Pi and -Pi treatments in the Student’s t-test (*: P < 0.05, **: 0.001 < P < 0.01, ***: P < 0.001). Figure S4 Physiological and biochemical levels in stylo roots response to low P stress. (a) Total P concentration of shoots and roots; (b) APase activity; (c) PAL activity; (d) total phenol content; (e) flavonoid content; (f) T-AOC activity; (g) malate concentration; (h) malate exudation rate. Stylo seedlings were precultured in hydroponics for 7 d with 250 μmol/L KH2PO4 and subsequently transferred into nutrient solution with (+Pi) or without (−Pi) 250 μmol/L KH2PO4 additions for 15 d. Each bar represents the mean of four replicates with SE (n = 4). Asterisks represent significant differences between +Pi and -Pi treatments in the Student’s t-test (*: P < 0.05, **: 0.001 < P < 0.01, ***: P < 0.001). P: phosphate, APase: acid phosphatase, T-AOC: total antioxidant capacity, PAL: phenylalanine ammonia lyase, DW: dry weight, FW: fresh weight. Figure S5 Phylogenetic analysis of stylo expansin proteins with other expansin proteins in plants. At: Arabidopsis thaliana; Gm: Glycine max; Ta: Triticum aestivum; Sg: Stylosanthes guianensis. EXPA: α-expansin; EXLA: expansin-like A; EXPB: β-expansin; EXLB: expansin-like B. Figure S6 Analysis of stylo root metabolites under two P treatments. (a) Principal component (PC) scores of metabolic the first two variances in stylo roots (n = 3). Stylo seedlings were grown in +Pi (250 μmol/L KH2PO4, triangles) and -Pi (0 μmol/L KH2PO4, circles) nutrient solutions for 15 d. The confidence level in the grey confidence circle is 95%. (b) Clustered heatmap of differentially accumulated metabolites (DAMs) in stylo roots at low P stress. Individual metabolites are represented by rows and nutritional status are represented by columns. Heatmap visualization of metabolites is based on standardized transformation (Z score) of metabolite concentrations. Figure S7 Expression analysis of SgPAPs and SgRNSs genes in response to Pi starvation. Expression levels of five SgPAPs and four SgRNSs were detected in stylo roots with (+Pi) or without (−Pi) 250 μmol/L KH2PO4 treatments for 15 d. Each bar represents the mean of four replicates with SE (n = 4). Asterisks indicate significant differences between +Pi and -Pi treatments in the Student’s t-test (*: P < 0.05, **: 0.001 < P < 0.01, ***: P < 0.001), ns indicates no significant difference. Figure S8 Circle graph of seven subcategory flavonoid metabolites in P deficient stylo roots. Digital scale represents the number of corresponding metabolites. Up-regulated or down-regulated metabolites were screened according to the criterion of fold changes ≥2.0 or ≤ 0.5, and VIP ≥1.0 (n = 3).