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Summary: 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. Significance Statement: Although the dogma of plant flavonoids proposes that labile plant flavonoids carbocations (PFCs) exist to associate with the biosynthesis of proanthocyanidins (PAs), supportive evidence is lacking. Data from biochemical analysis, isotope labeling, genetic analysis, metabolic engineering and metabolic profiling demonstrate that PFCs are involved in the biosynthesis of PAs. [ABSTRACT FROM AUTHOR]

Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a- overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a - and CsMYB5e -overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis . [ABSTRACT FROM AUTHOR]

The 2-oxoglutarate-dependent dioxygenase (2-OGD) superfamily is one of the largest protein families in plants. The main oxidation reactions they catalyze in plants are hydroxylation, desaturation, demethylation, epimerization, and halogenation. Four members of the 2-OGD superfamily, i.e., flavonone 3β-hydroxylase (F3H), flavones synthase I (FNS I), flavonol synthase (FLS), and anthocyanidin synthase (ANS)/leucoanthocyanidin dioxygenase (LDOX), are present in the flavonoid pathway, catalyzing hydroxylation and desaturation reactions. In this review, we summarize the recent research progress on these proteins, from the discovery of their enzymatic activity, to their functional verification, to the analysis of the response they mediate in plants towards adversity. Substrate diversity analysis indicated that F3H, FNS Ⅰ, ANS/LDOX, and FLS perform their respective dominant functions in the flavonoid pathway, despite the presence of functional redundancy among them. The phylogenetic tree classified two types of FNS Ⅰ, one mainly performing FNS activity, and the other, a new type of FNS present in angiosperms, mainly involved in C-5 hydroxylation of SA. Additionally, a new class of LDOXs is highlighted, which can catalyze the conversion of (+)-catechin to cyanidin, further influencing the starter and extension unit composition of proanthocyanidins (PAs). The systematical description of the functional diversity and evolutionary relationship among these enzymes can facilitate the understanding of their impacts on plant metabolism. On the other hand, it provides molecular genetic evidence of the chemical evolution of flavonoids from lower to higher plants, promoting plant adaptation to harsh environments. [ABSTRACT FROM AUTHOR]