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Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins
- Source :
- The Plant Journal. 101:18-36
- Publication Year :
- 2019
- Publisher :
- Wiley, 2019.
-
Abstract
- The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.
- Subjects :
- 0106 biological sciences
0301 basic medicine
Flavonoid
Ether
Plant Science
Biology
01 natural sciences
Camellia sinensis
Anthocyanidin reductase
Metabolic engineering
03 medical and health sciences
chemistry.chemical_compound
Biosynthesis
Gene Expression Regulation, Plant
Genetics
NADH, NADPH Oxidoreductases
Proanthocyanidins
Procyanidin B1
Procyanidin B2
Plant Proteins
Flavonoids
chemistry.chemical_classification
food and beverages
Leucoanthocyanidin reductase
Cell Biology
030104 developmental biology
chemistry
Biochemistry
010606 plant biology & botany
Subjects
Details
- ISSN :
- 1365313X and 09607412
- Volume :
- 101
- Database :
- OpenAIRE
- Journal :
- The Plant Journal
- Accession number :
- edsair.doi.dedup.....4a033c4555385e3f81d4791beb916866