Structural analysis of two distinct dihydroflavonol 4-reductases in Gerbera Hybrids

Dihydroflavonol 4-reductase (DFR) is a key enzyme within anthocyanin biosynthesis known for its distinct substrate specificity found in various plant species (1,2). Based in the findings of enzymatic studies and in vivo inhibition experiments in cyanidin accumulating genotype “Clivia” two DFRs with different substrate preference were postulated. Cloning approaches with petals of “Clivia” led to the identification of a second DFR sequence sharing around 96% identity to the previously cloned DFR from pelargonidin accumulating variety “Regina” (1,3). The obtained recombinant protein shows a higher preference for dihydroquercetin (DHQ) while dihydrokaempferol (DHK) was still converted to leucopelargondin (LPg) to a certain extend. However, the overall ratio showed a clear preference for the cyaniding branch of the pathway. To shed light on this biochemical aspect of the anthocyanins biosynthetic pathway, we have attempted an in silico structure-based approach aiming to relate the specific amino acid differences in the DFRs of Gerbera “Regina” and “Clivia” varieties, with their molecular structures and enzyme selectivity data. The DFR models have been built using the crystal structure of the DFR from Vitis vinifera4 (PDB ID 2C29) as a template. We suggest that a residue belonging to the “specificity loop” located near the substrate “binding site” confers the observed substrate-binding specificity. Namely, Gly135 (Gerbera “Regina” DFR) or Val135 (Gerbera “Clivia” DFR) respectively, is likely to unlock or lock the orientation of the conserved residue Asn134 that in turn is engaged in hydrogen bonding interactions with 4’ OH’ of ring B of DHK or both 3’ OH’ and 4’ OH’ of ring B of DHQ. Interestingly the side chain orientation of Val135 (Gerbera “Clivia” DFR) is restricted and stabilized by hydrophobic interactions with the conserved residues Ile154 and Phe165

Flavonoid metabolons in Gerbera hybrida

The formation of enzyme complexes, metabolons, and the channeling of intermediates of secondary metabolism has been discussed for at least 40 years (1). Metabolons and channeling enable plants to perform a highly effective synthesis of specific natural products without or with reduced metabolic interference and to avoid the accumulation of toxic intermediates (2). Despite a long tradition of the concept, precise examples of complete metabolons are scarce. For the anthocyanin pathway, soluble enzymes of the pathway have been shown to interact in yeast cells (3), and to associate with membranes in plant cells (4), the latter presumably through interactions with the membrane anchored P450 hydroxylases of the pathway. The ornamental plant Gerbera hybrida contains flavones, flavonols and anthocyanins in its petals, the exact composition reflecting the color of each gerbera cultivar. Orange, pink and red cultivars contain 4’ hydroxylated pelargonidin derivatives, while magenta and purplish cultivars are rich in 3’, 4’ hydroxylated cyanidin derivatives. Remarkably, 3’, 4’ hydroxylated flavones and flavonols (luteolin and quercetin) can be found in cultivars which completely lack cyanidin. The gerbera cultivar Terra Regina, with orange pelargonidin containing petals, starts to synthesize cyanidin when overexpressing the transcriptional regulator encoding GMYB10 gene, but not in cells where pelargonidin accumulates. These observations are indicative of metabolon control of flavonoid biosynthesis in gerbera petals. We have developed deep transcriptome data using Sanger, 454 and Illumina sequencing of different cultivars of gerbera (5 and unpublished). Assembly of the reads indicates that gerbera expresses small gene families for some of the flavonoid biosynthesis genes (e.g., PAL, 4CL, CHS and CHI) and single genes for others (e.g., DFR and F3’H). We are interested if the corresponding isoenzymes have different roles in biosynthesis of the array of flavonoids present in gerbera, particularly if they are able to form metabolons of different composition and biosynthetic capabilities. In order to map their interaction properties, we are running an all-against-all assay in yeast cells using vectors designed for membrane proteins

Anthocyanin biosynthesis in gerbera cultivars Estelle and Ivory

The acyanic cultivar Ivory is a sport of the pelargonidin containing pink cultivar Estelle, i.e., it originates from an acyanic branch of Estelle. To elucidate the flower pigments missing in Ivory, we collected samples from different developmental stages of Estelle and Ivory whole flowers and epidermi for anthocyanin analysis using Ultra-performance liquid chromatography tandem mass-spectrometry (UPLCMS/MS) and for RNA sequencing using the Illumina method. According to UPLC-MS/MS, we observed that Ivory lacked pelargonidin but had higher amounts of kaempferol glycosides. Apigenin glycosides were at similar levels as in Estelle. According to EST and Illumina sequencing data, we perceived that all anthocyanin biosynthesis genes were expressed similarly in the two cultivars. However, we found two allelic pairs (i.e., two loci) of the Dihydroflavonol 4-reductase (DFR) gene in Estelle and Ivory, and one of them carried in Ivory a single base mutation leading to amino acid change in the substrate binding sit

Two distinct dihydroflavonol 4-reductases involved in anthocyanin biosynthesis in Gerbera Hybrids

Gerbera hybrids are important cut flowers sold worldwide. Beside carotenoids in yellow inflorescenses, they are known to synthesize various anthocyanins giving orange, red and pink colors. Different varieties accumulating either pelargonidin (Pg) or cyanidin (Cy) derivatives or even both together have been described. Dihydroflavonol 4-reductase (DFR) is known as the key enzyme in anthocyanin biosynthesis and a gene coding for the respective protein was cloned and functionally characterized in vitro and in planta from the pink variety “Regina” (1, 2). From these studies it became apparent that the recombinant protein accepts both, dihydrokaempferol (DHK) and dihydroquercetin (DHQ), as substrates enabling the synthesis of Pg and Cy derivatives, respectively. The availability of DHQ depends on the activity of flavonoid 3’-hydroxylase (F3’H), a membrane bound cytochrome P450 protein, which was also cloned and characterized from the same variety (3). Moreover, the genetics of this step is also described. However, in vivo inhibition of F3’H by applying the specific inhibitor tetcyclacis resulted in white segments of the Cy-line “Clivia” which led to the assumption that the DFR in this specific variety is not able to convert DHK into the precursors of Pg-derivatives and therefore has different catalytical characteristics compared to the “Regina” DFR

Anthocyanin biosynthesis in gerbera cultivars ‘Estelle’ and its acyanic sport, ‘Ivory’

Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet color. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. ‘Ivory’, a cultivar with white petals, is a sport of the pelargonidin containing pink cultivar ‘Estelle’, i.e., it originates from an acyanic branch of ‘Estelle’. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to ‘Estelle’ with the functional allele GDFR1-2, ‘Ivory’ carries a mutation in this gene that results in an inactive enzyme. Interestingly, ‘Ivory’ also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin containing variety ‘President’. Pelargonidin containing cultivars do not react to the flavonoid 3’-hydroxylase inhibitor tetcyclacis, but cyanidin containing cultivars lose their color, instead of starting to synthesize pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of ‘Ivory’ are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier though