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.
Highlights
Chinese cabbage (Brassica rapa L. subsp. pekinensis) is an important vegetable mainly cultivated and consumed in East Asia
Like that of the early flavonoid biosynthesis genes (EBGs), expression levels of the late flavonoid biosynthesis genes (LBGs) peaked at 45 days after sowing (DAS) and decreased, consistent with the drastic increase in cyanidin content observed in 8267 at the later growth stage (Figure 3B). These results indicate that the expression patterns of BrPAL1.2 and the flavonoid biosynthetic genes correlated with the differences in flavonoid and anthocyanin accumulation in the two varieties; the expression of BrF3′H corresponds to the abundant content of 3′-hydroxylated flavonoids in 8267 leaves
These findings suggest that flavonol synthase (FLS) activity plays an important role in the pathway for both varieties, whereas the combined activities of anthocyanin biosynthetic enzymes and flavonoid 3′-hydroxylase (F3′H) are unique to the purple 8267 variety
Summary
Chinese cabbage (Brassica rapa L. subsp. pekinensis) is an important vegetable mainly cultivated and consumed in East Asia. Pekinensis) is an important vegetable mainly cultivated and consumed in East Asia This vegetable is highly nutritious and beneficial to human health due to its abundant content of phytochemicals such as glucosinolates, phenolic acids, carotenoids, and flavonoids (Šamec et al, 2011; Park et al, 2019), which commonly. A cross between green Chinese cabbage and turnip Purple Chinese cabbages were developed by crossing green Chinese cabbage with red bok choy Rubra; Lee et al, 2018) Metabolic profiling of these hybrid purple Chinese cabbages showed increased phenolic acid and flavonol (kaempferol, quercetin, and isorhamnetin) contents in leaves. 3′-hydroxylated flavonoids, such as quercetin and cyanidin, are the primary forms of flavonols and anthocyanidins, respectively, in purple Chinese cabbage (Jiang et al, 2013; He et al, 2016; Lee et al, 2018)
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