Fig. 1 Pathways for the biosynthesis of N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA) in potato tuber tissue. PAL, phenylalanine ammonia-lyase; C3′H, 5-O-(4-hydroxycinnamoyl) quinate 3′-hydroxylase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamic acid:CoA ligase; THT, tyramine hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase; CQT, quinate hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase. From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |

Fig. 2 Time course for the accumulation of p-CO (closed circles) and CGA (open circles) in wound-healing potato tuber tissue. At time –24, potato tuber disks were prepared and incubated at 18°C under dark conditions. The results are expressed as the mean of triplicate experiments ±SD. L is the straight line that passes through the two points on the CGA concentration curve at –6 h and +6 h. The slope of line L was defined as a constant v for non-linear regression analysis of CGA. From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |

Fig. 3 Schematic models of linear metabolic pathways Fig. 3 Schematic models of linear metabolic pathways. Symbols, X, Y and Z represent three metabolites in the pathway. J indicates the metabolic flux of each step. (A) A steady-state pathway. The flux from X to Y (defined as J) is equal to that from Y to Z. (B) A pathway in which the pool size of Y changes dynamically. The flux from X to Y (defined as J_in) is not equal to that from Y to Z (J_out). From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |

Fig. 4 Simplified pathways of p-CO and CGA biosynthesis for the analysis of metabolic flux using isotope-labeled phenylalanine. The four or five reaction steps in the pathway from phenylalanine to p-CO or CGA (Fig. 1) are simplified to one step by assuming that the levels of these intermediates are low enough. Based on the pathway, J_in and J_out of each metabolite were determined by metabolic flux analysis. The metabolic fate of CGA is not well understood. From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |

Fig. 5 Time course for the isotope abundance of l-phenyl-d₅-alanine (Phe*) in potato tuber tissue. At time 0, a 10 µl aliquot of 10 mM Phe* solution was applied to the tissue (~0.2 g) and incubated for a set amount of time. Following the removal of excess Phe* by thorough washing with distilled water, Phe and Phe* in the tissues were extracted and analyzed by LC-MS. The results are expressed as the mean of triplicate experiments ±SD. From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |

Fig. 6 The procedure for the analysis of metabolic flux of p-CO and CGA in wound-healing potato tuber tissue. (A) A typical HPLC chromatogram of potato extracts using UV detection at 280 nm. The separation of major metabolites was accomplished in 5 min. (B) SIM chromatograms of same samples with LC-MS (APCI positive ion mode) analysis. The protonated molecule [M+H]⁺ (m/z 355.1) and the ion formed by loss of water from the protonated molecule [M–H₂O+H]⁺ (m/z 282.1) were observed as the molecular-related ions in the case of CGA and p-CO, respectively. (C) SIM chromatograms of isotope-labeled and non-labeled p-CO in the potato extracts at 0, 0.5, 2 h after treatment with Phe* solution. Isotope-labeled p-CO (p-CO*) was observed at m/z 286.1 that was increased four mass units from the non-labeled p-CO (m/z 282.1). (D) Time course for the isotope abundance of p-CO (closed circles) and CGA (open circles) in wound-healing potato tuber tissue. Following the determination of the peak area of isotope-labeled (A*) and non-labeled (A) metabolite from the SIM chromatogram of LC-MS analysis, isotope abundance (A*/(A + A*)) was calculated. The results are expressed as the mean of triplicate experiments ±SD. The fitting curves of equation (7) are also shown. From: Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy Plant Cell Physiol. 2003;44(5):510-517. doi:10.1093/pcp/pcg063 Plant Cell Physiol |