Frommann, Jana-Freja: Untargeted metabolic fingerprinting of a loss-of-function and an overexpression mutant reveals involvement in flavonol and pyranoanthocyanidin glycosylation [...]. 2021
Inhalt
- Statement of authorship
- Abstract
- List of Abbreviations
- Introduction
- Plant secondary metabolites
- Phenolic compounds
- Flavonoids
- Flavonols
- Flavanols
- Anthocyanidins
- Glycosylation of flavonoids
- Functional characterization of unknown genes
- LC-MS based metabolite profiling
- Motivation and aim of this study
- Material and Methods
- Plant material
- Growth conditions
- Seed sterilization
- Genomic DNA extraction from plant material
- Loss-of-function T-DNA insertion line characterization
- Database derived expression prediction
- RNA isolation
- cDNA synthesis
- Semi-quantitative reverse transcriptase PCR
- Quantitative real time PCR
- Primer design
- Generation of loss-of-function double T-DNA insertion mutants
- Cloning of genomic DNA
- Cloning of genomic complementation constructs
- Transformation of electrocompetent E. coli TOP10 cells
- Plasmid isolation
- Enzymatic restriction of plasmids
- Gel electrophoresis
- Metabolite analysis
- Results
- Identification and characterization of T-DNA insertions in chosen bglu lines
- Differential gene expression of BGLU1, BGLU3, BGLU4 and BGLU5 between mutant lines and Arabidopsis wild type
- Candidate product and substrate features
- Identification of the main BGLU1 candidate product feature
- Identification of BGLU3 candidate product and substrate features
- Candidate product feature 866.4164 m/z at 11.75 min and candidate substrate feature 704.3635 m/z at 13.11 min
- Candidate product feature 773.2116 m/z at 10.51 min
- Further double-charged candidate product features
- Further single-charged candidate product features
- Candidate substrate feature 781.3270 m/z at 15.38 min
- Further candidate substrate features
- Identification of BGLU4 candidate product and substrate features
- Discussion
- BGLU1, BGLU3 and BGLU4 as possible GH1-type glycosyltransferases
- BGLU1 as possible flavonol GH1-type glycosyltransferase
- BGLU3 as a possible multifunctional flavonoid GH1-type glycosyltransferase
- BGLU4 as a possible flavonol GH1-type glycosyltransferase
- Further candidates of GH1-type glycosyltransferases in Arabidopsis
- The function of GH1-type GTs in plants
- Emergence of GH1-type GTs
- Flavanol–anthocyanidin, pyranoanthocyanidin and flavanol–pyranoanthocyanidin formation in Arabidopsis seeds
- The function of flavanol–anthocyanidin, pyranoanthocyanidin and flavanol–pyranoanthocyanidin in seeds
- Aglycones in seeds
- Future perspectives
- Conclusion
- References
- Supplement
- Sequences
- Sequence of transgene in 2x35S:BGLU1
- Sequence of transgene in 2x35S:BGLU3
- Sequence of transgene in 2x35S:BGLU4
- Sequence of pAC161
- Sequence of pROK2
- Acknowledgements