Generation and characterisation of an Arabidopsis thaliana f3h/fls1/ans triple mutant that accumulates eriodictyol derivatives.

BACKGROUND: Flavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxygenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each other's reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana, stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. RESULTS: To address this issue, we generated a f3h/fls1/ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h/fls1/ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h/fls1 and f3h/ans, was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta. Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3'-hydroxylase (F3'H) activity. CONCLUSIONS: We describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h/fls1/ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for the creation of novel food supplements.

Three R2R3-MYB transcription factors from banana (Musa acuminata) activate structural anthocyanin biosynthesis genes as part of an MBW complex.

OBJECTIVE: Bananas are one of the most popular fruits in the world, providing food security and employment opportunities in several developing countries. Increasing the anthocyanin content of banana fruit could improve the health-promoting properties. Anthocyanin biosynthesis is largely regulated at the transcriptional level. However, relatively little is known about the transcriptional activation of anthocyanin biosynthesis in banana. RESULTS: We analysed the regulatory activity of three Musa acuminata MYBs that were predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana. MaMYBA1, MaMYBA2 and MaMYBPA2 did not complement the anthocyanin-deficient phenotype of the Arabidopsis thaliana pap1/pap2 mutant. However, co-transfection experiments in A. thaliana protoplasts showed that MaMYBA1, MaMYBA2 and MaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, the so called MBW complex, resulting in the activation of the A. thaliana ANTHOCYANIDIN SYNTHASE and DIHYDROFLAVONOL 4-REDUCTASE promoters. The activation potential of MaMYBA1, MaMYBA2 and MaMYBPA2 was increased when combined with the monocot Zea mays bHLH ZmR instead of the dicot AtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. It will also facilitate research towards increased anthocyanin content in banana and other monocot crops.

Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

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

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.

Genome Sequencing of Musa acuminata Dwarf Cavendish Reveals a Duplication of a Large Segment of Chromosome 2.

Different Musa species, subspecies, and cultivars are currently investigated to reveal their genomic diversity. Here, we compare the genome sequence of one of the commercially most important cultivars, Musa acuminata Dwarf Cavendish, against the Pahang reference genome assembly. Numerous small sequence variants were detected and the ploidy of the cultivar presented here was determined as triploid based on sequence variant frequencies. Illumina sequence data also revealed a duplication of a large segment on the long arm of chromosome 2 in the Dwarf Cavendish genome. Comparison against previously sequenced cultivars provided evidence that this duplication is unique to Dwarf Cavendish. Although no functional relevance of this duplication was identified, this example shows the potential of plants to tolerate such aneuploidies.