Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation
Abstract
:1. Introduction
2. Results
2.1. Temporal Effect of Methyl Jasmonate on Intracellular Metabolites in C. tinctorius Cells
2.1.1. Promoting Effect on Chlorogenic Acid Accumulation
2.1.2. Metabolic Profile Changes of Primary Metabolites
2.2. Overall Transcriptional Alteration of C. tinctorius Cells in Response to MeJA Treatment
2.2.1. Results of Gene Annotation and New Gene Identification
2.2.2. Gene Differential Expression and Gene Set Enrichment Analysis
2.3. Transcriptional Differential Analysis of Endogenous Jasmonate Biosynthesis and Its Signaling Pathways in C. tinctorius Cells under MeJA Elicitation
2.4. Transcriptional Differential Analysis of CGA biosynthesis Pathway in MeJA-Induced C. tinctorius Cells
2.4.1. Transcriptional Changes in Shikimate Pathway
2.4.2. Transcriptional Changes in Phenylpropanoid Biosynthesis Pathway
2.4.3. Correlation Analysis between the Newly Annotated CtHCT Expression and CGA Accumulation in MeJA-Treated C. tinctorius Cells
2.5. Analysis of Bioinformatics, Subcellular Localization, and Transient Expression of the Newly Annotated and MeJA-Responsive CtHCT
2.6. In Vitro Catalytic Kinetic Analysis of the Recombinant CtHCT Protein
2.6.1. In Vitro Catalytic Activity Assay
2.6.2. Reaction Condition Optimization
2.6.3. Enzymatic Kinetic Analysis
2.7. Revealing the Catalytic Mechanism of the CtHCT Protein by Molecular Docking
3. Discussion
4. Materials and Methods
4.1. Product Induction of C. tinctorius Cells
4.2. Intracellular Metabolites Detection
4.3. Transcriptome Sequencing and Analysis
4.3.1. Transcriptome Sequencing
4.3.2. Gene Annotation and New Gene Identification
4.3.3. Gene Expression and Differential Analysis
4.4. Gene Expression Patterns Validation
4.5. Bioinformatics Analysis Methods
4.6. Subcellular Localization and Transient Transformation
4.7. Prokaryotic Expression and Purification of Protein
4.8. In Vitro Enzyme Activity Assays and Product Identification
4.9. Homologous Modeling and Molecular Docking
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Varying Substrate | Saturating Substrate | Km (µM) 1 | vmax (µm/min) 1 | vmax/Km (min−1) |
---|---|---|---|---|
p-Coumaroyl-CoA | Quinic acid | 9.41 | 10.02 | 1.06 |
Caffeoyl-CoA | Quinic acid | 32.61 | 1.71 | 0.05 |
Shikimic acid | p-coumaroyl-CoA | 18.38 | 25.12 | 1.37 |
Quinic acid | p-coumaroyl-CoA | 7.11 | 13.98 | 1.97 |
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Liu, Z.; Zhu, X.; Mohsin, A.; Sun, H.; Du, L.; Yin, Z.; Zhuang, Y.; Guo, M. Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation. Int. J. Mol. Sci. 2024, 25, 2710. https://doi.org/10.3390/ijms25052710
Liu Z, Zhu X, Mohsin A, Sun H, Du L, Yin Z, Zhuang Y, Guo M. Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation. International Journal of Molecular Sciences. 2024; 25(5):2710. https://doi.org/10.3390/ijms25052710
Chicago/Turabian StyleLiu, Zebo, Xiaofeng Zhu, Ali Mohsin, Huijie Sun, Linxiao Du, Zhongping Yin, Yingping Zhuang, and Meijin Guo. 2024. "Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation" International Journal of Molecular Sciences 25, no. 5: 2710. https://doi.org/10.3390/ijms25052710