Combining Stable Isotope Labeling and Candidate Substrate–Product Pair Networks Reveals Lignan, Oligolignol, and Chicoric Acid Biosynthesis in Flax Seedlings (Linum usitatissimum L.)
Abstract
1. Introduction
2. Results
2.1. LC–MS Analysis and Tracing SIL Moieties
2.2. Metabolite Identification Using Candidate Substrate–Product Pairs Network and CID Spectral Interpretation
2.3. Correlation and Time-Course Clustering
2.4. Quantification of Chicoric Acid
3. Discussion
3.1. SIL, MSn, and CSPP: A Synergistic Combination
3.2. Divergent Pathways of Coniferyl Alcohol Utilization During Flax Germination
3.3. Possible Alternative 3′-Hydroxylation Pathways in Flax Seedlings
3.4. Chicoric Acid
3.5. Benzoic Acids
4. Materials and Methods
4.1. Chemical Products
4.2. Plant Growth, Feeding, and Harvesting Conditions
4.3. Metabolite Extraction
4.4. Quantification of Chicoric Acid
4.5. Untargeted LC–MS Analysis of SIL Flax Seedlings
4.6. Tracing Labeled Metabolites
4.7. Mass Difference Networks
4.8. Time-Course Clustering
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Nr | m/z Difference | Conversion | Short | Elution Order | # CSPP |
---|---|---|---|---|---|
1 | 2.0157 | reduction | Red.neg | 1 | 7 |
2 | 14.0157 | methylation | Met | 2 | 2 |
3 | 15.9949 | oxygenation | Oxy | 1 | 10 |
4 | 18.0106 | hydration | Hydr | 1 | 3 |
5 | 30.0106 | methoxylation | Mox | 2 | 3 |
6 | 42.0106 | acetylation | Ace | 2 | 0 |
7 | 132.0060 | tartarate | Tar | 1 | 9 |
8 | 144.0423 | 1,6-anhydroglucose | anhydroGlc | 1 | 1 |
9 | 146.0579 | deoxyhexose | Rha | 1 | 0 |
10 | 156.0420 | shikimate | Shi | 1 | 0 |
11 | 162.0528 | hexose | Hex | 1 | 7 |
222.0740 | hexose and acetic acid adduct | Hex.AA | 1 | 12 | |
12 | 174.0530 | quinate | Qui | 1 | 0 |
13 | 192.0270 | hexaric acid | GlcA | 1 | 4 |
14 | 26.0157 | beta-oxydation | Box | 1 | 0 |
28.0224 | beta-oxydation.13C2 | Box.13C2 | 1 | 0 | |
15 | 104.0262 | benzoic acid | Ben | 2 | 0 |
104.0262 | benzoic acid.13C | Ben.13C | 2 | 0 | |
16 | 120.0211 | hydroxybenzoic acid | Phb | 2 | 0 |
121.0245 | hydroxybenzoic acid.13C1 | Phb.13C | 2 | 0 | |
17 | 146.0368 | coumarate | Cou | 2 | 8 |
149.0469 | coumarate.13C3 | Cou.13C3 | 2 | 8 | |
18 | 150.0317 | vanillic acid | Van | 2 | 0 |
151.0350 | vanillic acid.13C | Van.13C | 2 | 0 | |
19 | 162.0317 | caffeate | Caf | 2 | 4 |
165.0418 | caffeate.13C3 | Caf.13C3 | 2 | 7 | |
20 | 176.0473 | ferulate | Fer | 2 | 0 |
179.0574 | ferulate.13C3 | Fer.13C3 | 2 | 0 | |
21 | 178.0630 | condensed.guaiacyl | condGun | 2 | 2 |
181.0731 | condensed.guaiacyl.13C3 | condGun.13C3 | 2 | 2 | |
22 | 196.0736 | guaiacyl | Gun | 2 | 2 |
199.0837 | guaiacyl.13C3 | Gun.13C3 | 2 | 1 | |
23 | 206.0579 | sinapate | Sin | 2 | 0 |
209.0680 | sinapate.13C3 | Sin.13C3 | 2 | 0 | |
24 | 208.0735 | condensed.syringyl | condSun | 2 | 0 |
211.0836 | condensed.syringyl.13C3 | condSun.13C3 | 2 | 0 | |
25 | 226.0841 | syringyl | Sun | 2 | 0 |
229.0942 | syringyl.13C3 | Sun.13C3 | 2 | 0 | |
sum | 92 |
Nr | RT | Compound | Adduct | Observed m/z | Formula | Δ ppm | [Isotopologue] (Cluster) | CL |
---|---|---|---|---|---|---|---|---|
1 | 2 | Caffeoyl tartaric acid | [M−H]− | 311.0402 | C13H11O9 | −0.2 | [13C0] (28) | AN1 |
2 | 2.2 | Protocatechoyl glucose | [M−H]− | 315.0713 | C13H15O9 | −0.9 | [13C0] (29); [13C1] (29) | AN2 |
3 | 2.8 | p-Hydroxybenzoic acid glucoside | [M+Ac−H]− | 359.0977 | C15H19O10 | −0.3 | [13C0] (27); [13C1] (21) | AN1 |
4 | 3.1 | Gallic acid + glucose | [M−H]− | 331.0662 | C13H15O10 | −0.8 | [13C0] (30) | PU |
5 | 3.1 | Coumaroyl tartaric acid | [M−H]− | 295.0453 | C13H11O8 | −0.1 | [13C0] (5); [13C3] (6) | AN1 |
6 | 3.5 | Coumaroyl tartaric acid | [M−H]− | 295.0454 | C13H11O8 | −0.1 | [13C0] (7); [13C3] (6) | AN1 |
7 | 3.6 | Coumaroyl glucaric acid | [M−H]− | 355.0662 | C15H15O10 | −0.9 | [13C0] (13); [13C3] (14) | AN1 |
8 | 6.2 | Coumaric acid glucoside glucose ester | [M+Ac−H]− | 547.1659 | C23H31O15 | −0.8 | [13C0] (21); [13C3] (21) | AN2 |
9 | 6.2 | Caffeic acid glucoside | [M−H]− | 341.0871 | C15H17O9 | −0.5 | [13C0] (12); [13C3] (6) | AN1 |
10 | 6.9 | Coumaric acid glucoside | [M+Ac−H]− | 385.1131 | C17H21O10 | −1 | [13C0] (12); [13C3] (15) | AN2 |
11 | 8.5 | Ferulic acid glucoside | [M+Ac−H]− | 415.1234 | C18H23O11 | −1.5 | [13C0] (12); [13C3] (11) | AN1 |
12 | 8.5 | Dihydro-p-coumaroyl glucose | [M−H]− | 327.1077 | C15H19O8 | −0.9 | [13C0] (10); [13C3] (11) | AN2 |
13 | 8.8 | Coumaroyl glucose | [M−H]− | 325.0924 | C15H17O8 | 0.1 | [13C0] (8); [13C3] (9) | AN2 |
14 | 8.9 | Coniferin | [M+Ac−H]− | 401.1447 | C18H25O10 | −0.1 | [13C0] (16); [13C3] (2) | AN1 |
15 | 9 | Lariciresinol diglucoside | [M+Ac−H]− | 743.2749 | C34H47O18 | −1.8 | [13C0] (16); [13C3] (2); [13C6] (2) | PU |
16 | 9.4 | L-Chicoric acid | [M−H]− | 473.0718 | C22H17O12 | −0.4 | [13C0] (17); [13C3] (2); [13C6] (2) | ID |
17 | 9.6 | p-Coumaric acid | [M−H]− | 163.0398 | C9H7O3 | 1.8 | [13C0] (3); [13C3] (4) | ID |
18 | 10.3 | Coumaroyl caffeoyl tartaric acid | [M−H]− | 457.077 | C22H17O11 | −0.1 | [13C0] (17); [13C3] (11); [13C6] (11) | AN2 |
19 | 10.5 | Lariciresinol diglucoside | [M+Ac−H]− | 743.2744 | C34H47O18 | −2.5 | [13C0] (24); [13C3] (25); [13C6] (2) | PU |
20 | 10.7 | G(8-O-4)G-glucose | [M+Ac−H]− | 597.2165 | C28H37O14 | −4.0 | [13C0] (1); [13C3] (2); [13C6] (2) | AN1 |
21 | 11.4 | Pinoresinol diglucoside | [M+Ac−H]− | 741.2587 | C34H45O18 | −2.6 | [13C0] (23); [13C3] (15); [13C6] (14) | AN1 |
22 | 11.4 | Dicoumaroyl tartaric acid | [M−H]− | 441.0816 | C22H17O10 | −1.2 | [13C0] (17); [13C3] (11); [13C6] (11) | AN2 |
23 | 11.8 | Hydroxyphenyl acetic acid | [M−H]− | 151.0399 | C8H7O3 | 2.3 | [13C0] (7); [13C1] (7) | AN2 |
24 | 12.6 | Dicoumaroyl tartaric acid | [M−H]− | 441.0816 | C22H17O10 | −1.2 | [13C0] (18); [13C3] (19); [13C6] (19) | AN2 |
25 | 12.6 | Dehydrodiconiferyl alcohol glucoside | [M+Ac−H]− | 579.2074 | C28H35O13 | −0.7 | [13C0] (22); [13C3] (21); [13C6] (21) | AN1 |
26 | 12.9 | Lariciresinol glucoside | [M−H]− | 521.2015 | C26H33O11 | −1.6 | [13C0] (26); [13C3] (2); [13C6] (2) | AN1 |
27 | 13.9 | Pinoresinol glucoside | [M−H]− | 519.1865 | C26H31O11 | −0.2 | [13C0] (20); [13C3] (21); [13C6] (15) | AN1 |
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Thiombiano, B.; Mentag, A.; Paniez, M.; Roulard, R.; Marcelo, P.; Mesnard, F.; Dauwe, R. Combining Stable Isotope Labeling and Candidate Substrate–Product Pair Networks Reveals Lignan, Oligolignol, and Chicoric Acid Biosynthesis in Flax Seedlings (Linum usitatissimum L.). Plants 2025, 14, 2371. https://doi.org/10.3390/plants14152371
Thiombiano B, Mentag A, Paniez M, Roulard R, Marcelo P, Mesnard F, Dauwe R. Combining Stable Isotope Labeling and Candidate Substrate–Product Pair Networks Reveals Lignan, Oligolignol, and Chicoric Acid Biosynthesis in Flax Seedlings (Linum usitatissimum L.). Plants. 2025; 14(15):2371. https://doi.org/10.3390/plants14152371
Chicago/Turabian StyleThiombiano, Benjamin, Ahlam Mentag, Manon Paniez, Romain Roulard, Paulo Marcelo, François Mesnard, and Rebecca Dauwe. 2025. "Combining Stable Isotope Labeling and Candidate Substrate–Product Pair Networks Reveals Lignan, Oligolignol, and Chicoric Acid Biosynthesis in Flax Seedlings (Linum usitatissimum L.)" Plants 14, no. 15: 2371. https://doi.org/10.3390/plants14152371
APA StyleThiombiano, B., Mentag, A., Paniez, M., Roulard, R., Marcelo, P., Mesnard, F., & Dauwe, R. (2025). Combining Stable Isotope Labeling and Candidate Substrate–Product Pair Networks Reveals Lignan, Oligolignol, and Chicoric Acid Biosynthesis in Flax Seedlings (Linum usitatissimum L.). Plants, 14(15), 2371. https://doi.org/10.3390/plants14152371