Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field

Abstract

Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (−)-epigallocatechin-gallate [(−)-EGCG], (−)-epicatechin-gallate [(−)-ECG], (−)-epigallocatechin [(−)-EGC], (−)-epicatechin [(−)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (−)-EGCG, (−)-ECG, (−)-EGC, and (−)-EC accounted for 70–85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (−)-EC and (−)-Ca and (−)-EGC and (−)-GC, respectively, in which (−)-EC and (−)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (−)-EC and (−)-Ca and (−)-EGC and (−)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao’s leaves were produced from the ANR pathway.

Docking Characterization and in vitro Inhibitory Activity of Flavan-3-ols and Dimeric Proanthocyanidins Against the Main Protease Activity of SARS-Cov-2

“…Green tea (Zhao et al, 2017 ; Wang P. et al, 2020 ), two muscadine grape cultivar berries (including seeds and skin) (Yuzuak et al, 2018 ), and dark chocolate and cacao (Takahashi et al, 1999 ; Schewe et al, 2001 ) are rich in flavan-3-ol gallates and dimeric PAs. To understand whether these plant products could inhibit the M pro activity of SARS-Cov-2, we extracted flavan-3-ol gallates and dimeric PAs from the five types of materials and then completed in vitro assays.…”

“…At 10 μg/ml, the green tea extract tea completely inhibited the M pro activity ( Figure 7A ). In recent, we reported that green tea extracts contained a high content of EGCG, a relatively high content of ECG, and appropriate contents of dimeric procyanidin B1, B2, B3, and B4 (Zhao et al, 2017 ; Dai X. et al, 2020 ; Wang P. et al, 2020 ). Thus, the inhibitory activity of green tea extracts resulted from these compounds.…”

“…Flavan-3-ols and proanthocyanidins (PAs) are two groups of plant flavonoids ( Figure 2 ; Xie and Dixon, 2005 ). They commonly exist in fruits, food products, and beverages, such as grape (Iacopini et al, 2008 ; Zhu et al, 2014 ; Yuzuak et al, 2018 ; Rousserie et al, 2019 ), strawberry (Lopez-Serrano and Barcelo, 1997 ; Fossen et al, 2004 ; Fischer et al, 2014 ), persimmon (Akagi et al, 2009 ), cranberry (Foo et al, 2000a , b ), blueberry (Gu et al, 2002 ), cacao nuts (Murphy et al, 2003 ; Miller et al, 2009 ), chocolate (Schewe et al, 2002 ; Serafini et al, 2003 ), green tea (Liu et al, 2012 ; Zhao et al, 2017 ; Wang P. et al, 2020 ), and wines (Monagas et al, 2003 ). Common flavan-3-ol aglycones in these plant products include (–)-epicatechin (EC), (+)-catechin (CA), (–)-epigallocatechin (EGC), (+)-gallocatechin (GC), (–)-epiafzelechin (EAF), and (+)-afzelechin (AF) ( Figure 2 ; Xie and Dixon, 2005 ).…”

A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

“…This might be because CsWD40 promoted the regulation effects of CsMYB5e , resulting in higher PA biosynthesis than anthocyan in biosynthesis. In our previous studies, CsANR overexpression reduced anthocyanin production and promoted PA accumulation in the petals of tobacco plants [ 41 ]. In the Arabidopsis mutant of BAN (loss of function of ANR ), a high level of anthocyanin accumulated in the seed coat.…”

The genetic architecture of fruit colour in strawberry (Fragaria × ananassa) uncovers the predominant contribution of theF. vescasubgenome to anthocyanins and reveals underlying genetic variations

“…Indeed, we previously demonstrated (Fischer et al 2014) that ANR silencing in strawberry leads to the early accumulation of anthocyanins in the fruit, by re-routing the flux from flavan-3-ols to anthocyanins. At the opposite, Zhao et al (2017) recently showed that overexpression of tea ( Camellia sinensis ) ANR genes in tobacco effectively leads to a significant loss of flower red-pigmentation by reducing their anthocyanin content. These results further support the hypothesis that the increased expression of a LG3a-located ‘CF1116’ ANR homoeoallele from the F. vesca –derived subgenome is responsible for the increased accumulation of pelargonidin-3-glucoside that likely underlies the M3a colour mQTL.…”