Comprehensive Analysis of Metabolic Fluxes from Leucoanthocyanins to Anthocyanins and Proanthocyanidins (PAs)

Abstract

Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo. The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro. Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana, EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa. In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.

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“…In contrast, 1,3,7-trimethyluric acid and theacrine were not detected in the CK cultivars, which may be due to the lack of C8-oxidase. Caffeine was the dominant purine alkaloid in the JHKC individuals, which was consistent with findings concerning Niedu Kucha ( Camellia sinensis ) ( Wang, et al, 2020 ) but in contrast with findings concerning Yunnan Kucha [ Camellia kucha (Chang et Wang) Chang], whose predominant purine alkaloid was theacrine ( Yang et al, 2007 , Qin et al, 2020 ).…”

“…4 . Procyanidin B1 [EC-(4b,8)-C] and procyanidin B2 [EC-(4b,8)-EC] are EC-based PAs; procyanidin B3 [C-(4b,8)-C] and procyanidin B4 [C-(4b,8)-EC] are C-based PAs ( Wang, et al, 2020 ). Procyanidin B3 abundantly accumulated in the JHKC accessions, while procyanidin B1 and procyanidin B2 highly accumulated in the CK cultivars, as C was highly enriched in the JHKC accessions, while EC highly accumulated in the CK cultivars.…”

“…Among different tea plant species, theacrine was first detected in and isolated from Yunnan Kucha tea [ Camellia kucha (Chang et Wang) Chang] ( Ye, et al, 1999 ) and has subsequently been reported to exist in Puan tea ( Camellia sinensis var. puanensis Kurihara) ( Li, et al, 2017 ), Dayaoshan tea ( Camellia gymnogyna Chang) ( Teng, et al, 2020 ), Baiyacha ( Camellia gymnogyna Chang) ( Jin, Jiang, Yao, & Chen, 2020 ), and Niedu Kucha ( Camellia sinensis ) ( Wang, et al, 2020 ). In terms of its synthesis, theacrine is biosynthesized first through the conversion of caffeine to a 1,3,7-trimethyluric acid intermediate by a C8-oxidase, after which the intermediate is converted to theacrine by the N9-methyltransferase (CkTcS) ( Zheng et al, 2002 , Wang et al, 2020 , Zhang et al, 2020 ).…”

“…puanensis Kurihara) ( Li, et al, 2017 ), Dayaoshan tea ( Camellia gymnogyna Chang) ( Teng, et al, 2020 ), Baiyacha ( Camellia gymnogyna Chang) ( Jin, Jiang, Yao, & Chen, 2020 ), and Niedu Kucha ( Camellia sinensis ) ( Wang, et al, 2020 ). In terms of its synthesis, theacrine is biosynthesized first through the conversion of caffeine to a 1,3,7-trimethyluric acid intermediate by a C8-oxidase, after which the intermediate is converted to theacrine by the N9-methyltransferase (CkTcS) ( Zheng et al, 2002 , Wang et al, 2020 , Zhang et al, 2020 ). Among the 11 JHKC individuals evaluated in this study, 7 individuals contained theacrine, and the theacrine contents of the JHKC individuals ranged from 12.27 to 26.33 mg/g ( Fig.…”

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“…Leucoanthocyanidin is the common substrate for LAR and ANR and it is biosynthesized by converting dihydroflavonols into leucoanthocyanidins by dihydroflavonol 4-reductase (DFR). Additionally, before the catalytic action of ANR, the anthocyanidin synthase (ANS) must catalyze the conversion of leucoanthocyanidin into anthocyanidin (Wang et al, 2020).…”