Protein Expression of Flavonol Synthase from Fragrant Wild Cyclamen (Cyclamen Purpurascens)

Abstract

To aid the development of DNA markers and new flower-color mutants, we previously isolated and analyzed the flavonoid biosynthesis-related genes involved in flower coloration in fragrant wild cyclamen (Cyclamen purpurascens). Two flavonol synthase genes (CpurFLS1 and CpurFLS2) were subsequently isolated and CpurFLS2 found to be related to flower coloration. As a next step, in vitro observations of enzymatic activity are necessary; therefore, expression analysis of CpurFLS2 protein was conducted. In this study, we provide the optimal conditions for recombinant soluble CpurFLS2 protein production in Escherichia coli.

Keywords

Cyclamen, Flavanol synthase, Protein expression.

Introduction

Three cultivars of fragrant cyclamen are currently available, all of which were created by crossing the cyclamen cultivar Cyclamen persicum with the scented species C. purpurascens [1]. Our overall aim is to create new fragrant cultivars with novel petal coloration using ion-beam irradiation. Because ion-beam irradiation tends to cause large DNA rearrangement, PCR screening is useful in selecting desired mutants from a mutagenized cyclamen population before flowering. Clarification of the mechanism of flower coloration and identification of the genes involved in flower coloration in cyclamen are therefore necessary [2].

Cyclamen flower color is controlled by an accumulation of major plant pigment-related compounds known as flavonoids. The main flavonoids in C. purpurascens flowers are a single anthocyanin, malvidin 3,5-diglucoside (Mv3,5dG), and two flavanols, quercetin and kaempferol [3,4]. Anthocyanins are well-known plant pigments responsible for strong flower coloration. Numerous anthocyanin biosynthesis-related genes have been isolated from a number of plant species including cyclamen [5-8]. However, the gene(s) for Flavanol Synthase (FLS) in cyclamen has yet to be isolated. Flavanols occasionally modify flower color when combined with anthocyanins, a process known as co-pigmentation [9]. For example, reduced FLS leads to reddening of flower color in petunia [10]. Determining the combination of anthocyanin and flavanols is therefore important in understanding flower coloration. We recently isolated two FLS genes from C. purpurascens (CpurFLS1 and CpurFLS2), and in a complement experiment using an fls mutant of Arabidopsis thaliana, found that both genes function in flavonol synthase. Differential expression of these two genes was also revealed, with constitutive expression of CpurFLS1 in young petals and other organs (Figure 1). In contrast, CpurFLS2 expression was strong in young petals but weak in anthers, leaves and petioles. Moreover, no expression of CpurFLS2 was observed in open petals (Figure 1). These patterns suggest that CpurFLS1 and CpurFLS2 possess functional diversity, with a correlation between CpurFLS2 and flower coloration in C. purpurascens. As a next step, it is therefore important to determine the functional differences between these two genes, including substrate specificity. In this report, we therefore examine the optimal conditions for CpurFLS2 protein expression and purification [10].

Figure 1: Flower of Cyclamen purpurascens and the expression patterns of CpurFLS1 and CpurFLS2 shown as blue density. YP: Young Petal; OP: Open Petal; IA: Immature Anther.

Materials and Methods

Protein Expression and Purification

The full-length open reading frame (ORF) of CpurFLS2 cDNA (accession number: LC210073) was amplified by PCR using a forward primer with the NdeI site and reverse primer with the XhoI site. The amplified fragments were subcloned into the pGEM-T Easy vector (Promega) and digested with NdeI and XhoI. Then, the excised fragment was ligated into the NdeI–XhoI site of the His-tagged pET16b expression vector (Merck) to yield pET16b-CkmOMT2. The insertion was sequenced carefully using T7 primer sets to verify that no mutations occurred. To produce recombinant proteins with a His-tag at the N-terminus, the resulting plasmid pET16b-CkmOMT2 was used to transform Escherichia coli strain BL21 (DE3) (Merck). E. coli harboring pET16b-CkmOMT2 was cultivated in 2 ml of lysogeny broth (Difco) supplemented with 100 µgmL-1 ampicillin until reaching an OD₆₀₀ of 0.4–0.5. After addition of isopropyl ß-D-thiogalactopyranoside (IPTG) to a final concentration of 0–0.1 mM, the cells were further cultured at 20°C to 37°C for 616 h. They were then resuspended in FastBreakTM Cell Lysis Reagent (Promega) and the fusion protein purified using the HisLinkTM Spin Protein Purification System (Promega) according to the manufacturer’s protocol.

Results and Discussion

Our aim was to examine CpurFLS2 protein production in recombinant E. coli under various conditions. At an incubation temperature of 20°C, recombinant CpurFLS2 protein expression was observed after addition of 10 µM IPTG, with increasing expression under 50 µM IPTG (Figure 2). Furthermore, expression was higher with an incubation time of 8 h compared to 6 h (Figure 2). However, with an incubation time of more than 10 h, CpurFLS2 expression was the same as that under no IPTG. We subsequently adjusted the incubation temperature (25°C, 28°C, 30°C, and 37°C, respectively) and similarly analyzed the effect on expression. Under all conditions, an increase in CpurFLS2 protein was observed; however, in most cases, the protein was insoluble (data not shown). As a result, we determined the optimal conditions for recombinant soluble CpurFLS2 protein production in E. coli as 1) a final concentration of IPTG of 10 µM, 2) an incubation temperature of 20°C, and 3) an incubation time of 8 h.

Figure 2: Protein expression of recombinant CpurFLS2 under various culture conditions. The incubation temperature was kept constant at 20°C.

We subsequently attempted to purify the recombinant CpurFLS2 protein using a His-tagged expression vector. As a result, using the above optimal culture conditions, N-terminus His-tagged CpurFLS2 protein was successfully produced in recombinant E. coli. The isoelectric point of CpurFLS2 was 6.15 and the putative molecular weight was 37.7 kDa (data not shown). The molecular mass of the purified protein was consistent with the expected mass of the fusion protein including the additional His-tag of 2 kDa (Figure 3). These results suggested successful purification of recombinant soluble CpurFLS2. The next step is to carry out an enzymatic assay of CpurFLS2 to determine enzymatic activity and substrate specificity. Two FLS proteins were recently isolated from different colored onions, with only three amino acid differences between AcFLS-H6 (isolated from red onion) and AcFLSHRB (isolated from yellow onion). Despite the relatively high amino acid similarity, the catalytic efficiency of AcFLS-HRB was approximately twice that of AcFLS-H6 when dihydroflavonol was used as a substrate. Comparisons of the predicted amino acid sequences of CpurFLS1 and CpurFLS2 revealed approximately 50 differences (Figure 4), suggesting differences in enzymatic activity and/or substrate specificity. To examine enzymatic activity, we therefore expressed CpurFLS1 under the above culture conditions. As a result, strong expression of recombinant CpurFLS1 was observed similar to CpurFLS2; however, all were insoluble (data not shown).

Figure 3: Comparison of the deduced amino acid sequences of CpurFLS1 and CpurFLS2. Amino acid residues identical in the two sequences are indicated by a black background.

Figure 4: Purification of recombinant CpurFLS2. Lane M: Protein Marker; Lane 1: E. coli Lysate without IPTG; Lane 2: E. coli Lysate with 10 µM IPTG; Lane 3: The Purified Recombinant Protein. The purified CpurFLS2 recombinant protein is indicated by an arrowhead.

Conclusion

These results suggest that the optimal conditions for production of recombinant soluble protein differ between CpurFLS1 and CpurFLS2. Further clarification of the optimal conditions for expression of soluble recombinant CpurFLS1 are therefore necessary. We previously purified anthocyanin-O-methyltransferase (CkmOMT2) from the fragrant cyclamen cultivar ‘Kaori-nomai’ [11,12]. The optimal conditions for CkmOMT2 production were 1) a final concentration of IPTG of 100 µM, 2) an incubation temperature of 28°C, and 3) an incubation time of 18 h. Thus, despite the proteins being isolated from cyclamen, the optimal culture conditions for protein expression differed depending on the protein. In a future study, we will examine the question of why the protein expression conditions differ. These findings will aid research on flower coloration in cyclamen.

Acknowledgments

We are grateful to Prof. Masahide Ishikawa (Saitama Institute of Technology) for providing help and advice. Funding This study was funded by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (JP15K18641) awarded to Y. Akita.

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