Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

Abstract

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that the divergence of the expression patterns for FhDFR genes might be controlled at transcriptional level, as the expression of FhDFR1/FhDFR2 and FhDFR3 was controlled by a potential MBW regulatory complex with different activation efficiencies. Therefore, it can be concluded that the DFR-like genes from F. hybrida have diverged during evolution to play partially overlapping roles in the flavonoid biosynthesis, and the results will contribute to the study of evolution of DFR gene families in angiosperms, especially for monocot plants.

A Radish Basic Helix-Loop-Helix Transcription Factor, RsTT8 Acts a Positive Regulator for Anthocyanin Biosynthesis

“…In the transient assay, ectopic expression of RsMYB 1 in tobacco induced the high expression of NtAN1 gene that was overlapping the expression pattern of anthocyanin biosynthetic pathway genes (Figure 5 ). These results were consistent with previous work that bHLH and MYB proteins have essential roles in anthocyanin biosynthesis (Liu et al, 2016 ; Li et al, 2017 ). Interestingly, the transcript level of RsMYB1 and RsTTG1 was the highest, but that of RsTT8 was lower in white root flesh (D variety) as a similar result with white potato flesh (Liu et al, 2016 ).…”

Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins

“…Genetic transformation followed the floral dipping transformation protocol [36]. The growth of infected plants and seed harvesting followed the same protocol as previously reported [9,37]. T1 seeds were selected on PNS medium with 50 mg L −1 kanamycin to obtain T2 plants.…”

SNP in DFR1 Coding Sequence Is Tightly Associated with Anthocyanin Accumulation in Cabbage (B. oleracea var. capitata f. alba) at Low Temperature

“…Genetic and molecular characterization of DFR genes have been conducted in various plant species including alfalfa (Medicago truncatula) [36], trembling aspen (Populus tremuloides) [53], lotus (Lotus japonicus) [54], Saussurea medusa [55], Chinese cabbage [49], and Freesia hybrida [56]. In plants, the DFR gene generally exists as two or more copies, for example, two copies in maize (Zea mays) [57], alfalfa [36], poplar (Populus spp.)…”

“…In plants, the DFR gene generally exists as two or more copies, for example, two copies in maize (Zea mays) [57], alfalfa [36], poplar (Populus spp.) [58], and yam (Dioscorea alata) [27], three copies in morning glory (Ipomoea batatas) [59], wheat (Triticum aestivum) [60], onion (Allium cepa) [61], and ginkgo (Ginkgo biloba) [62], six copies in lotus [63], eight copies in Freesia hybrida [56], and twelve copies in Chinese cabbage [49]. Multiple copies of the DFR genes imply the functional diversification of DFR proteins for color determination, stress response, and other horticultural traits.…”