Introduction
Delphinium, a member of the Ranunculaceae family, grows naturally in the alpine areas of China, Central Asia, and the United States (US). It is valued in the market as a garden plant with blue flowers. In Europe and US, the large-flowered Delphinium elatum is popular, while in Japan and South Korea, the small spray-flowered D. grandiflorum is popular. Both species were bred to produce a wide variation of bluish-flowered cultivars. However, the reddish-flowered species D. nudicaule and D. cardinale are available in the market. Both species have not been improved, and there are few reddish-flowered cultivars. The accumulation of anthocyanins produces the flower color of Delphinium. The structure of the accumulated anthocyanins in deep blue flowers is highly complex and characteristic, leading to cyanodelphin and violdelphin. Our previous studies have identified anthocyanin modifying enzyme genes involved in synthesizing violdelphin1). It has also been shown that the flower color differs depending on the deficient mutant of the anthocyanin modifying enzyme gene. Thus, our research aims to investigate the genes involved in anthocyanin biosynthesis and modification in various Delphinium species and cultivars to produce novel flower colors. In Delphinium, the bluish flowers accumulate anthocyanins derived from delphinidin, while the reddish flowers accumulate anthocyanins derived from pelargonidin2). Previous studies have not identified any Delphinium species that can synthesize anthocyanins derived from cyanidins. Herein, we demonstrate a study that produced a novel reddish flower variety synthesizes cyanidin by hybridizing breeding3,4).
Materials and Methods
2.1 Plant materials
The seedlings of D. zalil and D. cardinale were obtained from Miyoshi & Co., Ltd. and cultivated in a greenhouse at the Research and Development Center of Miyoshi & Co., Ltd.
2.2 Heterologous expression of DzF3'H protein in yeast
The protein coding region of DzF3'H cDNA was amplified by polymerase chain reaction (PCR) using the primer set with a HindIII and XbaI site at each end. The amplicon was introduced into a pYES2 (Invitrogen, USA) expression vector with a URA3 selection marker, and the resultant pYES2-DzF3'H was transferred into Saccharomyces cerevisiae strain INVSc1.
2.3 Recombinant enzyme reaction
Crude protein extracts from yeast transformants were prepared by a Multi-beads shocker (Yasui Kikai Co., Japan) with glass beads (0.5 mm diameter) at 10 cycles of 2,500 rpm for 10 s. The cell debris was removed by centrifugation at 10,000 × g for 5 min at 4°C, and the supernatant was used as the crude protein extract in the enzyme assay. The F3′H enzyme reaction was performed for 2 h at 30°C, using 90 g crude protein extract. The protein extract was added to 60 nmol substrates and 100 nmol NADPH in a total volume of 100 L 0.1 M potassium phosphate buffer (pH 7.5).
2.4 Flavonoid profiles
Approximately 200 mg of sepals from fully-opened flowers were frozen in liquid nitrogen and ground into powder using a mortar and pestle. Subsequently, the powder was mixed in 100 μL of 80% methanol containing 0.1% trifluoroacetic acid. The cell debris was removed by centrifugation at 15,000 × g for 10 min, and the supernatant was High-performance liquid chromatography (HPLC) analyzed.
2.5 HPLC condition
The separations were carried out for 5 min using an HPLC-photodiode array detector system equipped with an ODS column (4.6 × 50 mm, COSMOSIL 5C18 -MS-II, Nacalai Tesque, Japan), and a linear gradient elution (1 mL·min−1) of 25%–50% methanol in 1.5% aqueous phosphoric acid. The sepal extract was separated by linear gradient elution at a flow rate of 1 mL·min−1 in 20%–80% methanol/1.5% aqueous phosphoric acid for 20 min and detected at 520 nm.
Results and Discussion
The analysis of flavonoids in sepals indicated that Delphinium zalil accumulates large amounts of quercetin 3-glucoside. In D. zalil, anthocyanin synthase expression is suppressed, resulting in a lack of anthocyanin synthesis and yellow colored flowers. For this reason, D. zalil has not been used for breeding and producing blue or red colored flowers. Quercetin is synthesized by reacting flavonoid 3'-hydroxylase (F3'H) using dihydrokaempherol and kaempherol as substrates. F3'H is an essential enzyme for the synthesis of cyanidin. In many delphiniums, F3'H is not expressed in the sepals; therefore, cyanidin cannot be synthesized in flowers. RT-PCR showed that DzF3'H is expressed in the sepal, so we investigated substrate specificity by heterologous expression in yeast. The results showed that the recombinant DzF3'H converted naringenin, apigenin, dihydrokaempherol, and kaempherol into eriodictyol, luteolin, dihydroquercetin, and quercetin (Table 1).
Table 1 Substrate preferences of recombinant DzF3’H.
| Substrate | Relative activity (%) |
|---|---|
| Naringenin | 56 |
| Apigenin | 68 |
| Dihydrokaempferol | 100 |
| Kaempherol | 57 |
The broad range of substrate specificities, and especially the intense activity toward dihydrokaempherol, suggested that introducing the DzF3'H gene into other delphiniums could lead to synthesizing cyanidin. Next, we cross-hybridized D. cardinale with D. zalil. D. cardinale lacked F3'5'H function and is known to accumulate large amounts of anthocyanins derived from pelargonidin, resulting in red solid colored flower. The flower color of F1 hybrids was shown to accumulate anthocyanins with cyanidin as the main structure (Fig.1). Also, the flower color was reddish purple, a color not seen in previous Delphinium varieties. The anthocyanin structure is presumed to be a cyanidin-based structure with violdelphin-type modifications.
Fig. 1 Anthocyanidin profile of F1 hybrid of D. zalil and D. cardinal.
Conclusion
Most nurseries are breeding varieties without using genetically modified plants due to consumer concerns about the safety of genetically modified plants and their impact on the environment. In our research, we selected Delphinium species for breeding based on the expression analysis of anthocyanin synthesis and modification enzymes, focusing on developing reddish flower colors. As a result, we succeeded in breeding hybrids with anthocyanin derived from cyanidin.