Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor

Abstract

Amaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

A Genome-Wide Identification Study Reveals That HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT Involved in Betalain Biosynthesis in Hylocereus

“…Betaxanthins were detected in yeast cultures after co-expression of HmoCYP76AD1 and HmoDODAα1 genes without feeding L-DOPA ( Figure 9 ), suggesting that HmoCYP76AD1 has tyrosine hydroxylase activity for converting tyrosine to L-DOPA. Similar findings were also reported in other studies [ 17 , 18 , 19 ]. Red pigmentation was detected after expression of HmoCYP76AD1 , HmoDODAα1 and HmocDOPA5GT in N. benthamiana and yeast culture ( Figure 8 and Figure 9 ), indicating that HmoCYP76AD1 catalyzes two succeeding steps in the pitaya betalain biosynthesis pathway to produce cyclo-DOPA.…”

“…Red pigmentation was detected after expression of HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT in N. benthamiana and yeast culture (Figures 8 and 9), indicating that HmoCYP76AD1 catalyzes two succeeding steps in the pitaya betalain biosynthesis pathway to produce cyclo-DOPA. Similar results were also found in other betalain-producing plant species such as B. vulgaris, C. quinoa, A. tricolor and M. jalapa [13,[16][17][18]22,23].…”

“…The functions of CYP76ADα and CYP76ADβ clade homologues have been previously reported in betalain-producing plant species [ 13 , 15 , 16 , 17 , 18 , 19 ]. In B. vulgaris , silencing CYP76AD1 alone blocked the production of betacyanins but not betaxanthins, suggesting that CYP76AD1 and CYP76AD6 catalyze the formation of L-DOPA from tyrosine.…”

“…Chenopodium quinoa [16] and Amaranthus tricolor [17]. The CYP76AD1-β genes (BvCYP76AD5, BvCYP76AD6 and MjCYP76AD15) encoding the enzyme convert tyrosine into L-DOPA which have been confirmed in M. jalapa and B. vulgaris [18,19].…”

“…Based on phylogenetic analyses of 100 Caryophyllales plants, CYP76AD1-like genes are classified into CYP76AD1-α, CYP76AD1-β and CYP76AD1-γ [ 14 ]. The functions of CYP76AD1-α genes ( BvCYP76AD1 , MjCYP76AD3 , CqCYP76AD1 and AtriCYP76AD1 ) encoding betalain biosynthesis enzymes have been elucidated in Beta vulgaris [ 13 ], Mirabilis jalapa [ 15 ], Chenopodium quinoa [ 16 ] and Amaranthus tricolor [ 17 ]. The CYP76AD1-β genes ( BvCYP76AD5 , BvCYP76AD6 and MjCYP76AD15 ) encoding the enzyme convert tyrosine into L-DOPA which have been confirmed in M. jalapa and B. vulgaris [ 18 , 19 ].…”