Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage

Abstract

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

Tartary buckwheat FtF3′H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant

“…These results are consistent with the reports in Chinese Cabbage ( Brassica rapa L. subsp. pekinensis; He et al, 2020a , b , Park et al, 2021 ), O. sativa ( Shih et al, 2008 ), and Paeonia suffruticosa ( Shih et al, 2006 ; Zhang et al, 2020 ; Nitarska et al, 2021 ). In our experiments, FtF3′H1 increased the accumulation of anthocyanin, which may due to the higher expression abundance of FtF3′H1 than FtFLS s or FtF3′H1 has a stronger substrate preference for anthocyanin branch.…”

“…It is a brilliant cereal with edible and medicinal characteristics. Enriched flavonoids provide the pharmacological activity for TB ( Li et al, 2012b ; Dong et al, 2020 ; Park et al, 2021 ). As a focus in the secondary metabolism of plants, flavonoids not only endow various colors including red, blue, and purple as pigmentation resources in plants, but also are associated with health as active compounds in vegetables and fruits ( Grotewold, 2006 ).…”

“…For example, F3′H overproduction induced the F3′H-and F3′5′H-deficient pale-pink petunia to produce fuchsia flowers due to the accumulated anthocyanidins ( Tanaka, 2006 ). In Actinidia ( Peng et al, 2019 ), Arachis hypogaea ( Xue et al, 2021 ), Brassica rapa ( Park et al, 2021 ), and other plants, the expression of F3′H was strongly correlated with the content of different flavonoids, respectively. Obviously, F3′H is involved in multiple actions and has a significant effect on flavonoid metabolism in plants.…”

Identification of Hydroxylation Enzymes and the Metabolic Analysis of Dihydromyricetin Synthesis in Ampelopsis grossedentata

“…Exploring its metabolic pathway and related genes for application in foods [ 18 , 26 ] and medicines [ 17 , 19 , 20 , 27 ] was of great significance. Many studies have been performed on Ag F3H, Ag F3′H, and Ag F3′5′H in different plants, such as Reaumuria trigyna [ 28 ], Brassica rapa [ 23 ], Brunfelsia acuminata [ 12 ], and Solanum lycopersicum [ 29 ]. However, the function of Ag F3′H, Ag F3′5′H, and Ag CPR in A. grossedentata has not been reported.…”

“…Li and Yu performed solid preliminary studies; however, all key genes were predicted instead of verified in vitro or in vivo. The key genes related to DHM biosynthesis, F3′H and F3′5′H, are members of the cytochrome P450 enzyme family and required the participation of cytochrome P450 reductase (CPR) and NADPH [ 23 ]. The P450 and CPR genes need anchoring to the endoplasmic reticulum membrane surface to perform their functions.…”