Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis

Abstract

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

Keywords: anthocyanidin synthase; banana; flavanone 3-hydroxylases; flavonol synthase; specialised metabolites.

Figure 1

Schematic, simplified illustration of the core flavonoid aglycon biosynthesis pathway in plants. Chalcone synthase, the first committed enzyme in flavonoid biosynthesis, connects a CoA ester and malonyl CoA, forming a chalcone. Chalcone isomerase introduces the heterocyclic ring. The resulting flavanone is converted to dihydroflavonol by flavanone 3-hydroxylase (F3H) or to flavones by flavone synthase (FNSI or FNSII). Dihydroflavonols are converted to flavonols by flavonol synthase (FLS). Alternatively, dihydroflavonol 4-reductase can reduce dihydroflavonols to the corresponding leucoanthocyanidin, which is converted to anthocyanidin by the activity of anthocyanidin synthase (ANS). Flavonoid 3' hydroxylase hydroxylates 3' position of the B-ring using different flavonoid substrates. 2-oxoglutarate-dependant oxygenases are given in bold underlined. Enzymes in the focus of this work are highlighted by grey boxes.

Figure 2

Rooted approximately maximum-likelihood (ML) phylogenetic tree of 2-ODDs involved in flavonoid biosynthesis. ODDs from banana are given in blue, 39 enzymes with proven F3H, FNSI, FLS or ANS activity were included. Different grey scales indicate F3H, FLS, ANS and two evolutionary FNSI clades. AtGA3ox1 (gibberellin 3 beta-hydroxylase1) was used as an outgroup. Branch points to DOXC28 and DOXC47 classes are marked with black arrowheads.

Figure 3

MusaF3H1 and MusaF3H2 are functional F3Hs. (A) HPTLC analysis of F3H bioconversion assays using extracts from Escherichia coli expressing recombinant MusaF3H1 or MusaF3H2. The substrate naringenin (N) and the product dihydrokaempferol (DHK) were used as standards. AtF3H was used as a positive control. AtMYB12 was used as a negative control. (B) Analysis of tt6-2 mutant seedlings complemented with MusaF3H1 and MusaF3H2. Col-0 wild type and tt6-2 were used as controls. Representative pictures of DPBA-stained seedlings under UV light are given. Yellow fluorescence indicates flavonol glycoside accumulation. The different numbers indicate individual transgenic lines. The scale bar indicates 0.5 mm.

Figure 4

MusaFLS1 and MusaFLS3 are functional FLSs. (A) HPTLC analysis of a FLS bioconversion assays using extracts from E. coli expressing recombinant MusaFLS1 or MusaFLS3. The F3H substrate naringenin (N), the FLS substrate DHK and the product kaempferol (K) were used as standards. AtFLS1 served as a positive control and AtMYB12 was used as a negative control. (B) Flavonol glycoside accumulation in MusaFLS-complemented ans/fls1-2 seedlings analysed by HPTLC analysis. Col-0, Nö-0 (both wild type) and ans/fls1-2 were used as controls. Bright green spots belong to derivatives of kaempferol, orange spots are derivatives of quercetin and faint blue shows sinapate derivatives. Dark green and yellow spots indicate DHK and DHQ, respectively. G, glucose; K, kaempferol; Q, quercetin; and R, rhamnose. (C) Representative pictures of anthocyanin (red) accumulation in 6-day-old MusaFLS-complemented ans/fls1-2 seedlings growing on 4% sucrose. The scale bar indicates 0.5 mm.

Figure 5

MusaANS is a functional anthocyanin synthase. Analysis of ans/fls1-2 double mutant seedlings complemented with 2x35S-driven MusaANS demonstrate in planta ANS functionality of MusaANS by anthocyanin accumulation. The different numbers indicate individual transgenic lines. (A,B) Sucrose induced anthocyanin accumulation in 6-day-old Arabidopsis thaliana seedlings. (A) Representative pictures of seedlings (the scale bar indicates 0.5 mm) and (B) corresponding relative anthocyanin content. Error bars indicate the standard error for three independent measurements. (C) MusaANS does not show in planta FLS activity. Flavonol glycoside accumulation in MusaANS-complemented ans/fls1-2 seedlings analysed by HPTLC analysis. Col-0, Nö-0 (both wild type) and ans/fls1-2 were used as controls. Bright green spots belong to derivatives of kaempferol, orange spots are derivatives of quercetin and faint blue shows sinapate derivatives. Dark green and yellow spots indicate DHK and DHQ, respectively. G, glucose; K, kaempferol; Q, quercetin; and R, rhamnose.