Metabolomic Profiling Identifies Key Metabolites and Defense Pathways in Rlm1-Mediated Blackleg Resistance in Canola
Abstract
1. Introduction
2. Results
2.1. Multivariate Analysis of Metabolomic Data
2.2. Univariate Analysis of Metabolomic Data
2.3. DAMs in Relation to Inoculation and Resistance
2.4. Prominent DAMs and Their Related Pathways
2.5. Metabolites/Pathways Potentially Related to Rlm1-Mediated Resistance
2.5.1. Lysine Metabolism and Degradation
2.5.2. Defense Signaling Molecules
2.5.3. Antimicrobial Metabolites
2.5.4. Amino Acid and Secondary Metabolite Pathways
2.5.5. Scopoletin/Isoscopoletin Biosynthesis
2.5.6. Redox Metabolism (GSH/GSSG)
2.5.7. Flavonoid-Related Pathways
2.6. Validating DAM Candidates for Their Potential Roles in Rlm1-Mediated Resistance
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Pathogen Isolates
4.2. Plant Inoculation, Infection Assessment, and Leaf-Tissue Sampling
4.3. Sample Preparation for Metabolomic Analysis Using CIL LC–MS
4.4. Metabolome Quantification
4.5. LC−MS Analysis
4.6. LC–MS Raw Data Extraction and Processing
4.7. Validating the Potential Involvement of Selected Metabolites in Resistance
4.7.1. Chemical (Metabolite) Preparation
4.7.2. Application of Metabolites
4.7.3. Inoculation and Infection Assessment
4.8. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Pathways | Total Compounds | Hits | Raw p | Impact |
---|---|---|---|---|
Flavone and flavonol biosynthesis | 10 | 4 | 5.2649 × 10−5 | 0.5 |
Isoquinoline alkaloid biosynthesis | 6 | 1 | 0.013614 | 0.5 |
Arginine and proline metabolism | 32 | 3 | 0.11938 | 0.32738 |
Biosynthesis of various plant secondary metabolites | 29 | 1 | 0.24485 | 0.24 |
Glycine, serine and threonine metabolism | 33 | 2 | 0.018356 | 0.22375 |
Lysine biosynthesis | 9 | 1 | 0.000103 | 0.16216 |
Arginine biosynthesis | 18 | 1 | 0.17605 | 0.13981 |
Tryptophan metabolism | 29 | 1 | 0.010177 | 0.10687 |
Glyoxylate and dicarboxylate metabolism | 29 | 1 | 0.11936 | 0.10147 |
Tyrosine metabolism | 17 | 1 | 0.013614 | 0.10056 |
Phenylpropanoid biosynthesis | 43 | 2 | 0.00128 | 0.09634 |
Glutathione metabolism | 26 | 1 | 0.11936 | 0.07114 |
Pyrimidine metabolism | 41 | 1 | 0.021007 | 0.02929 |
Purine metabolism | 73 | 3 | 0.000316 | 0.02344 |
Phenylalanine, tyrosine, and tryptophan biosynthesis | 22 | 2 | 0.011081 | 0.02002 |
Flavonoid biosynthesis | 47 | 2 | 4.2424 × 10−5 | 0.00338 |
Cysteine and methionine metabolism | 47 | 1 | 0.000562 | 0.00265 |
Lipoic acid metabolism | 24 | 1 | 0.11936 | 0.0016 |
D-Amino acid metabolism | 7 | 1 | 0.000103 | 0 |
Indole alkaloid biosynthesis | 4 | 1 | 0.010177 | 0 |
Glucosinolate biosynthesis | 65 | 1 | 0.010177 | 0 |
Ubiquinone and other terpenoid-quinone biosynthesis | 47 | 1 | 0.013614 | 0 |
Anthocyanin biosynthesis | 11 | 1 | 0.014562 | 0 |
Lysine degradation | 20 | 1 | 0.038832 | 0 |
Thiamine metabolism | 22 | 1 | 0.11936 | 0 |
Cyanoamino acid metabolism | 29 | 2 | 0.16748 | 0 |
Pathways | Total Compounds | Hits | Raw p | Impact |
---|---|---|---|---|
Taurine and hypotaurine metabolism | 5 | 2 | 0.004092 | 1 |
Glutathione metabolism | 26 | 5 | 0.000843 | 0.51637 |
Isoquinoline alkaloid biosynthesis | 6 | 3 | 0.000207 | 0.5 |
Tyrosine metabolism | 17 | 4 | 0.001364 | 0.32961 |
Glycine, serine and threonine metabolism | 33 | 1 | 7.41 × 10−5 | 0.22375 |
Arginine biosynthesis | 18 | 2 | 0.020249 | 0.17088 |
Lysine degradation | 20 | 3 | 5.38 × 10−5 | 0.16667 |
Lysine biosynthesis | 9 | 1 | 0.000818 | 0.16216 |
Flavone and flavonol biosynthesis | 10 | 2 | 0.00029 | 0.15 |
Arginine and proline metabolism | 32 | 3 | 0.040159 | 0.14584 |
Butanoate metabolism | 17 | 1 | 0.13621 | 0.13636 |
Cysteine and methionine metabolism | 47 | 2 | 0.000311 | 0.13181 |
Alanine, aspartate, and glutamate metabolism | 22 | 1 | 0.13621 | 0.1295 |
Purine metabolism | 73 | 3 | 0.001022 | 0.10374 |
Glyoxylate and dicarboxylate metabolism | 29 | 1 | 7.41 × 10−5 | 0.10147 |
Phenylpropanoid biosynthesis | 43 | 1 | 0.017894 | 0.05935 |
Pyrimidine metabolism | 41 | 1 | 0.000913 | 0.02929 |
Folate biosynthesis | 31 | 1 | 0.39026 | 0.02624 |
Porphyrin metabolism | 48 | 1 | 0.001198 | 0.02261 |
Ubiquinone and other terpenoid-quinone biosynthesis | 47 | 2 | 4.84 × 10−5 | 0.02209 |
Phenylalanine, tyrosine, and tryptophan biosynthesis | 22 | 2 | 4.84 × 10−5 | 0.02002 |
Tryptophan metabolism | 29 | 3 | 0.000722 | 0.01527 |
Lipoic acid metabolism | 24 | 1 | 7.41 × 10−5 | 0.0016 |
Thiamine metabolism | 22 | 1 | 7.41 × 10−5 | 0 |
Cyanoamino acid metabolism | 29 | 2 | 8.89 × 10−5 | 0 |
Glucosinolate biosynthesis | 65 | 3 | 0.000128 | 0 |
Flavonoid biosynthesis | 47 | 1 | 0.000193 | 0 |
Biosynthesis of various plant secondary metabolites | 29 | 1 | 0.000248 | 0 |
Valine, leucine, and isoleucine degradation | 37 | 1 | 0.000662 | 0 |
Valine, leucine, and isoleucine biosynthesis | 22 | 1 | 0.000662 | 0 |
D-Amino acid metabolism | 7 | 1 | 0.000818 | 0 |
Tropane, piperidine, and pyridine alkaloid biosynthesis | 9 | 2 | 0.003453 | 0 |
Anthocyanin biosynthesis | 11 | 1 | 0.012109 | 0 |
Zeatin biosynthesis | 21 | 1 | 0.044967 | 0 |
Pathways | Total Compounds | Hits | Raw p | Impact |
---|---|---|---|---|
Taurine and hypotaurine metabolism | 5 | 3 | 0.003645 | 1 |
Phenylalanine metabolism | 12 | 1 | 0.000113 | 0.42308 |
Glutathione metabolism | 26 | 3 | 9.13 × 10−5 | 0.40276 |
Tyrosine metabolism | 17 | 5 | 0.000141 | 0.39665 |
Phenylpropanoid biosynthesis | 43 | 8 | 9.23 × 10−5 | 0.28583 |
Ubiquinone and other terpenoid-quinone biosynthesis | 47 | 2 | 0.000443 | 0.1998 |
beta-Alanine metabolism | 18 | 2 | 0.022568 | 0.19444 |
Lysine degradation | 20 | 3 | 0.000743 | 0.16667 |
Lysine biosynthesis | 9 | 1 | 0.000192 | 0.16216 |
Arginine and proline metabolism | 32 | 3 | 3.65 × 10−6 | 0.15774 |
Butanoate metabolism | 17 | 1 | 0.000299 | 0.13636 |
Alanine, aspartate and glutamate metabolism | 22 | 1 | 0.000299 | 0.1295 |
Purine metabolism | 73 | 3 | 0.003473 | 0.09255 |
Phenylalanine, tyrosine and tryptophan biosynthesis | 22 | 3 | 1.11 × 10−5 | 0.09159 |
Arginine biosynthesis | 18 | 1 | 0.004743 | 0.08641 |
Pyrimidine metabolism | 41 | 2 | 0.002791 | 0.07198 |
Flavonoid biosynthesis | 47 | 5 | 0.00363 | 0.06956 |
Cysteine and methionine metabolism | 47 | 5 | 0.000575 | 0.05644 |
Glucosinolate biosynthesis | 65 | 3 | 1.36 × 10−6 | 0.04236 |
Tryptophan metabolism | 29 | 4 | 4.93 × 10−5 | 0.03054 |
Pantothenate and CoA biosynthesis | 25 | 1 | 0.16968 | 0.02796 |
Porphyrin metabolism | 48 | 1 | 0.013372 | 0.02261 |
Flavone and flavonol biosynthesis | 10 | 2 | 6.24 × 10−5 | 0 |
Cyanoamino acid metabolism | 29 | 1 | 0.000113 | 0 |
Tropane, piperidine and pyridine alkaloid biosynthesis | 9 | 3 | 0.000161 | 0 |
D-Amino acid metabolism | 7 | 1 | 0.000192 | 0 |
Anthocyanin biosynthesis | 11 | 2 | 0.00027 | 0 |
Glycine, serine and threonine metabolism | 33 | 1 | 0.002717 | 0 |
Zeatin biosynthesis | 21 | 1 | 0.005646 | 0 |
Isoquinoline alkaloid biosynthesis | 6 | 2 | 0.006486 | 0 |
Common Name | Abbreviation | Chemical Name | Mol. Formula | Concentration 2 | Supplier |
---|---|---|---|---|---|
Pipecolic acid | PA | Piperidine-2-carboxylic acid | C6H11NO2 | 40 mM | Tokyo Chemical Industry (TCI) (Portland, OR, USA) |
Salicylic acid (sodium salt) | SA | Sodium 2-hydroxybenzoate | C7H5NaO3 | 1 mM | Thermo Fisher (Ottawa, ON, Canada) |
Gentisic acid (sodium salt hydrate) | GA | 2,5-Dihydroxybenzoic acid sodium salt | C7H5O4Na | 10 mM | Sigma Aldrich (MilliporeSigma Canada Ltd., Oakville, ON, Canada) |
Glutathione | GSH | γ-L-glutamyl-L-cysteinylglycine | C6H11NO2 | 20 mM | Thermo Fisher |
Lysine | Lys | (S)-2,6-Diaminocaproic acid | C6H14N2O2 | 10 mM | Sigma Aldrich |
Diaminopimelic acid | DAP | 2,6-Diaminopimelic acid | C7H14N2O4 | 30 mM | Sigma Aldrich |
Ferulic acid | FA | Trans-ferulic acid | C10H10O4 | 1 mM 3 | Sigma Aldrich |
Caffeic acid | CFA | (E)-3-(3,4-dihydroxyphenyl) prop-2-enoic acid | C9H8O4 | 10 mM 4 | Sigma Aldrich |
Benzoic acid | BA | Benzoic acid | C7H6O2 | 10 mM 5 | Thermo Fisher |
Piperonylic acid | PipA | 1,3-benzodioxole-5-carboxylic acid | C8H6O4 | 3 mM 6 | Sigma Aldrich |
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Zhu, X.; Gao, P.; Zhao, S.; Luo, X.; Li, L.; Peng, G. Metabolomic Profiling Identifies Key Metabolites and Defense Pathways in Rlm1-Mediated Blackleg Resistance in Canola. Int. J. Mol. Sci. 2025, 26, 5627. https://doi.org/10.3390/ijms26125627
Zhu X, Gao P, Zhao S, Luo X, Li L, Peng G. Metabolomic Profiling Identifies Key Metabolites and Defense Pathways in Rlm1-Mediated Blackleg Resistance in Canola. International Journal of Molecular Sciences. 2025; 26(12):5627. https://doi.org/10.3390/ijms26125627
Chicago/Turabian StyleZhu, Xiaohan, Peng Gao, Shuang Zhao, Xian Luo, Liang Li, and Gary Peng. 2025. "Metabolomic Profiling Identifies Key Metabolites and Defense Pathways in Rlm1-Mediated Blackleg Resistance in Canola" International Journal of Molecular Sciences 26, no. 12: 5627. https://doi.org/10.3390/ijms26125627
APA StyleZhu, X., Gao, P., Zhao, S., Luo, X., Li, L., & Peng, G. (2025). Metabolomic Profiling Identifies Key Metabolites and Defense Pathways in Rlm1-Mediated Blackleg Resistance in Canola. International Journal of Molecular Sciences, 26(12), 5627. https://doi.org/10.3390/ijms26125627