Elucidation of Low-temperature Regulated Flavone Synthesis in Dahlia Variabilis and its Effects on Flower Color (2025)

Abstract

Dahlia (Dahlia variabilis) flower colors are diverse and are determined by the accumulation of flavonoids. Cultivars with dark red flowers accumulate more anthocyanins in their petals. Flower color changes such as color fading often occur in some cultivars. In this study, low minimum temperature regulated flower color fading and flavonoid synthesis in dahlia ‘Nessho’ were investigated. The pigment contents and expression levels of flavonoid biosynthesis genes were investigated in detail under several growing environments in which color fading occurs. Flavones accumulate more in color-faded orange flowers than in dark red ray florets. The expression analysis of the anthocyanin synthesis pathway genes indicated that the upregulation of flavone synthase (DvFNS) gene expression correlated with the high accumulation of flavones in color-faded petals. DvFNS expression was also detected in young leaves, and the expression level was higher in winter than in summer. Seasonal changes in DvFNS expression in young leaves significantly correlated with color fading in petals. The change in DvFNS expression in young unexpanded leaves of relatively high-sensitive plants was significantly higher than that of low-sensitive plants before and after treatment under inductive conditions. In conclusion, low-temperature-inducible changes in the flavonoid accumulation in petals was suggested to reflect a change in DvFNS expression occurring in the meristem prior to flower bud formation. This temporal DvFNS expression in young unexpanded leaves of ‘Nessho’ dahlia could be an insight for the selection and breeding of non-color fading plants.

Introduction

Dahlias (Dahlia variabilis) are popular floricultural crops that are used as cut flowers or garden plants. Various cultivars with widely diverse flower colors have been bred, i.e., black, purple, orange, pink, red, white, and yellow, while other cultivars exhibit variegated or bicolor phenotypes. The flower colors result from flavonoid accumulation, mainly consisting of anthocyanins, butein, and flavone glycosides and their derivatives (Halbwirth etal., 2008; Harborne etal., 1990; Nordström and Swain, 1953; Ohno etal., 2011a). The flavonoid biosynthetic genes in dahlia have been largely elucidated (Deguchi etal., 2013; Ohno etal., 2011a, b, 2013, 2022).

Seasonal change in flower color has been reported in several plant species, including dahlia. Environmental factors such as temperature play key roles in the regulation of anthocyanin biosynthesis and pigment accumulation in plants (Carbone etal., 2009; Jaakola, 2013). Fukuta and Nakayama (2008) reported that the colored area on the petals of the eustoma (Eustoma grandiflorum) bicolor cultivar remained for longer in the winter while the white areas were greatly reduced. Suzuki etal. (2010) reported that color fading occurred in dahlia when the minimum temperature was 10°C. The instability of dahlia flower colors has been the focus of several studies (Deguchi etal., 2013, 2015; Ohno etal., 2011a, b, 2016, 2022). In these studies, post-transcriptional gene silencing (PTGS) of chalcone synthase (DvCHS) or type II (cytochrome P450) flavone synthase (DvFNS) genes were shown to be involved in the instability of the flower color in dahlia. The latter was induced by tobacco streak virus (TSV_dahlia) infection and resulted in color-changing occurrence in dark red colored dahlia cultivars. Further, Ohno etal. (2018) reported that the instability of the flower color in the dahlia bicolor cultivar ‘Yuino’ reflected the PTGS induction for DvCHS at the whole plant level.

‘Nessho’ is a dahlia cultivar with dark red flowers that exhibits color fading from autumn through spring in response to a minimum temperature under 10°C (Fig.1), at which time the ray florets turn orange (Okada etal., 2020; Suzuki etal., 2010). Color fading progresses quantitatively along with decrease in minimum temperature. The incipient stage of color-fading occurrence is slight discoloration on the whole ray floret on a capitulum in mid-autumn (Fig.1b). After that, partial color-fading in ray florets occurs until early winter (Fig.1c, d), and finally, all ray florets on a capitulum are an orange color from mid-winter (Fig.1e). The L*a*b* chromaticity values range from L*28.1 ± 0.4, a*41.2 ± 0.3, b* 40.8 ± 0.4 for dark red flowers to L* 42.4 ± 0.7, a* 40.0 ± 0.9, b* 46.4 ± 0.5 for completely color-faded orange flowers (Okada etal., 2020). Deguchi etal. (2013) suggested that the color changing occurrence of dark red dahlia is the result of a change in flavonoid composition, particularly an increase in flavone contents and decrease in anthocyanin contents. If the color-fading phenomenon in ‘Nessho’ is caused by the same mechanism, we may be able to elucidate the mechanism of low-temperature regulated flavone synthesis. In chrysanthemums, prolonged high temperature inhibits flavone biosynthesis by directly regulating CmFNS and anthocyanin by suppressing the expression of CmCHS, CmDFR, CmANS, and CmUFGT (Zhou etal., 2021). However, to the best of our knowledge, no other study has reported on low-temperature regulated flavone synthesis.

Deguchi etal. (2015) reported that TSV_dahlia infection caused color-changing occurrences in darker red dahlia cultivars; however, there is no report indicating any virus involvement in color-fading occurrences in ‘Nessho’ dahlia. Further, it is reported that the ‘Nessho’ cultivar consists of some plants with relatively high sensitivity to low-temperature conditions (RH-sensitive plants) (Okada etal., 2020). RH-sensitive plants quickly respond to changes in the minimum temperature under 10°C and exhibit color fading in their ray florets; thus, they are a good model for analyzing the regulation of the flavone synthesis pathway.

In the present study, we attempted to elucidate the regulation of the flavone synthesis pathway under low-temperature conditions that cause flower color fading in dahlia using vegetatively propagated RH-sensitive plants. We identified the possible flavonoid synthesis pathway gene for color-fading occurrence under low-temperature conditions. Further, we sought to identify whether or not the seasonal regulation in the flavonoid synthesis pathway spread over the whole plant. We examined this by analyzing the expression of the potential causal flavonoid synthesis pathway gene in leaves from summer through winter. Our results show a relationship between DvFNS gene expression in leaves, color-fading occurrence in ray florets, and the minimum temperature under experimental conditions.

Materials and Methods

Plant materials

A dahlia cultivar plant with relatively high sensitivity to low-temperature conditions (RH-sensitive plant) was vegetatively propagated, and the plantlets were maintained and used for the subsequent experiments according to our previous report (Okada etal., 2018). Petals and leaves were collected from greenhouse-grown plants in the experimental field of Shinshu University (Minamiminowa, Japan) and used in the subsequent analyses. Additionally, relatively low sensitivity plants (RL-sensitive plants) and RH-sensitive plants were selected and grown in an Okayama University experimental field according to our previous study (Okada etal., 2020). Plants used for gene expression analysis were grown in an incubator under controlled conditions.

Pigment analysis

The extraction of flavonoids was done as previously described by Deguchi etal. (2013). The setup of the High-performance liquid chromatography (HPLC) system, pump, detector, column, gradient program, and solvents was previously described by Hamauzu etal. (2018). The detection wavelength was 350nm for flavones and 530nm for anthocyanins. To make a standard curve, 1mg of known pelargonidin, cyanidin, apigenin, and luteolin were eluted in 600μL buffer (10% hydrochloric acid in 50% methanol) and five serial dilutions were made. Twenty microliters of each dilution were injected into the HPLC and pigment amounts were calculated from peak areas. Amounts of pigments were quantified as peak area-equivalents from 100mg ray floret fresh weight. Three to four dark red ray florets and color-faded orange ray florets were sampled from distinct capitula and analyzed as biological replicates. Comparison of flavonoid amounts-equivalents of each flavonoid from the samples was carried out by analysis of variance (ANOVA), with pairwise multiple comparisons for the Tukey-Kramer test.

Expression analysis for flavonoid synthesis pathway genes

Expression analysis of flavonoid synthesis pathway genes was conducted using real-time reverse transcription (RT)-PCR. Unexpanded ray florets were sampled from distinct capitula. Developmental stages of the ray florets were defined as follows: stage 1, 10mm length ray floret; stage 2, 20mm length ray floret; stage 3, 30mm length ray floret. Three red ray florets and four color-faded orange ray florets were analyzed as biological replicates. Total RNA (> 200 nucleotides) was extracted using NucleoSpin miRNA (Takara Bio Inc., Shiga, Japan), and purified with a high-salt solution for precipitation (Takara Bio Inc.) two times. The extracted total RNA was reverse-transcribed with ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan), and 1μL of RT product was used as a template for real-time RT-PCR. Real-time RT-PCR was performed with THUNDERBIRD^® SYBR qPCR Mix (TOYOBO) according to the manufacturer’s instructions using a Dice^® Real-Time System Thermal Cycler (Takara Bio Inc.). The primers used were as reported by Ohno etal. (2011b). The PCR program was set at 95°C for 1min, followed by 40 cycles of 95°C for 15s, 60°C for 1min, and subsequent dissociation steps. Expression profiles of D.variabilis Anthocyanin synthase, DvANS, Dihydroflavonol 4-reductase, DvDFR, Flavone synthase DvFNS, Flavanone 3-hydroxylase, DvF3H, and Flavonoid 3'-hydroxylase DvF3'H were analyzed. DvActin was used as an internal standard. Additionally, DvFNS expression was analyzed using ray florets that exhibited variegated coloration. Since ‘Nessho’ capitula often produce red and orange variegated ray florets, each red and orange area was separated from four distinct ray florets. Total RNA was extracted as mentioned above and used for the DvFNS expression analysis using the same primer sets and real-time RT-PCR conditions.

DvFNS expression in leaves and its relationship with color fading occurrence in ray florets

To confirm whether the seasonal changes in DvFNS expression occur only in ray florets but not in leaves, we analyzed the DvFNS expression in leaves at different maturation stages. The optimum leaf maturation stage for DvFNS expression analysis was decided using leaf blades sampled from RH-sensitive plants in February, when they produced completely color-faded orange ray florets. Unexpanded leaves and leaves on the 1^st to 4^th internodes were sampled from four distinct plants as biological replicates (Fig.4a). Total RNA was extracted with a Get pure RNA Kit (Dojindo, Kumamoto, Japan). Extracted total RNA was reverse transcribed as described above, and 1μL of RT product was used as a template for real-time RT-PCR. Real-time RT-PCR was performed with THUNDERBIRD^® SYBR qPCR Mix (TOYOBO) according to the manufacturer’s instructions using a StepOne^TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used were as reported by Ohno etal. (2011b). The PCR conditions and internal standard were as previously described.

DvFNS expression in unexpanded leaves was analyzed from weeks 32 to 53 using 210 vegetatively propagated RH-sensitive plants. Total RNA extraction, reverse transcription, and real-time RT-PCR were conducted as previously described. Three to six unexpanded leaves sampled from distinct plants were analyzed as biological replicates. In conjunction with the DvFNS expression analysis, the color fading occurrence in ray florets was analyzed for the same plants. The extent of color fading on the nearly fully open terminal capitulum was evaluated according to the color fading index (CFI) (Fig.1; Okada etal., 2020): CFI 0, color fading was not observed; CFI 1, slight color fading was observed, but the color change was not completely orange; CFI 2, color-faded ray florets exhibited a completely orange color, but red ray florets dominated the capitulum; CFI 3, color-faded ray florets exhibited orange color and orange ray florets dominated the capitulum; CFI 4, all ray florets were completely orange. Pearson’s correlation coefficients were analyzed for DvFNS expression level in leaves, CFI values and average weekly minimum temperature.

Confirmation of the difference in DvFNS expression levels between RH-sensitive plants and RL-sensitive plants

DvFNS expressions in young unexpanded leaves were compared between eight strains of RL-sensitive plants and three strains of RH-sensitive plants. Plants were approximately 20cm in height with more than three internodes and were pinched on the second internode to allow new lateral shoot development. After pinching, the plants were transferred to an incubator set at acclimation conditions (25°C/20°C day and night temperature and 14 hours day length) to homogenize the DvFNS gene expression levels in all plants. Young unexpanded leaves were sampled 14 days after the onset of the acclimation condition. The conditions of the incubator were changed to inductive conditions of 20°C/9°C day and night temperatures and 10 hours day length, thus mimicking winter conditions to induce DvFNS gene expression. The second sampling was done 14 days after the onset of the inductive condition. The samples were used to obtain total RNAs. Total RNA extraction, reverse transcription, and real-time RT-PCR were conducted as described above.

Results

Pigment analysis of ray florets

To clarify the difference in pigments between color-faded orange ray florets and original dark red ray florets, aglycones of anthocyanins and flavones were extracted from ray florets and analyzed by HPLC. The four analyzed aglycones (two anthocyanidins and two flavones) were detected differently in color-faded orange and dark red ray florets. The color-faded orange ray florets accumulated 7 to 10-fold higher amounts of flavones (apigenin and luteolin glycosides) than the dark red florets (Fig.2). The dark red ray florets accumulated up to 9-fold higher amounts of anthocyanins (cyanidin and pelargonidin) contents compared to the color-faded orange ray florets. The accumulation of anthocyanidins in the ray florets was inversely proportional to that of the flavone glycosides.

Quantitative real-time RT-PCR analysis of flavonoid biosynthetic pathway genes

To determine the regulation of flavonoid biosynthesis genes in the ‘Nessho’ dahlia cultivar, the level of expression of major structural genes in the anthocyanin synthetic pathway was analyzed. The expression levels of anthocyanin genes DvANS, DvDFR, DvF3H, and DvF3'H did not differ between the color-faded orange ray florets and dark red ray florets. However, higher DvFNS expression was detected in color-faded orange ray florets at all analyzed developmental stages (Fig.3a). There was a distinct difference in the level of DvFNS expression between the three developmental stages with the expression levels being greatest in stage 3 ray florets. DvFLS expression was undetectable in all three developmental stages in both the dark red and the orange-colored ray florets. The expression levels of DvANS, DvDFR, DvF3H, and DvF3'H were not different across the ray florets’ developmental stages, although they were consistently higher in the 3^rd stage.

Comparison of the gene expression profiles for red and orange variegated ray florets showed that the expression level of DvFNS was significantly different between the red and the orange regions (P < 0.05). There were also small, but significant, differences in the expression of DvCHS1, DvCHS2, and DvCHI (Fig.3b).

DvFNS expression in leaves and its relationship with color-fading occurrences in ray florets

Leaves at five maturation stages; unexpanded leaves, leaves at the 1^st, 2^nd, 3^rd, and 4^th nodes of RH-sensitive plants were used to evaluate whether DvFNS expression change occurred in the flower stage and also in the leaves in response to minimum low-temperature. DvFNS expression levels were greatest in the young unexpanded leaves (Fig.4a). The expression levels declined up to 5-fold and more in the leaves above the 1^st, 2^nd, 3^rd, and 4^th nodes. DvFNS expression levels in young unexpanded leaves of RH-sensitive plants were compared between the winter (February) and summer (June) seasons. There was higher DvFNS expression during winter compared to summer for the same plants as shown in Figure4b.

Further, DvFNS expression in young unexpanded leaves was analyzed for 210 RH-sensitive plants and compared to the CFI of the capitulum of the same plants from August to December, 2015. During the months of August and September, DvFNS expression in young unexpanded leaves was low, as was the corresponding CFI (Fig.5). From October through November, DvFNS expression levels gradually increased, as did the CFI values. In December, the DvFNS expression levels were highest, and this correlated with high CFIs (3 to 4), indicating extreme color fading from the dark red color. The pattern of expression correlated with the color fading indices.

To ascertain the relationship between temperature, DvFNS expression and seasonal flower fading in ‘Nessho’, a correlation analysis was done. As the level of DvFNS expression increased, the flower CFI increased (Fig.6a), indicating a positive correlation (r = 0.775) between DvFNS expression and degree of color fading. The correlation between the flower color fading index and average weekly minimum temperature indicated an inverse relationship such that as the average weekly minimum temperature increased, the color fading occurrence decreased (Fig.6b). Similarly, as the average weekly minimum temperature increased, there was a reduced level of DvFNS gene expression, and hence a negative regression (R² = 0.744; Fig.6c).

A comparison between the change in DvFNS expression levels in young unexpanded leaves of RL- and RH-sensitive plants before and after inductive conditions indicated a significant difference at the 95% confidence level. The relative expression levels in RH-sensitive plants were higher than in RL-sensitive plants (Fig.7).

Discussion

Flower color change in response to changes in environmental conditions, mainly temperature, light intensity and daylength is a common phenomenon reported in a variety of plants. Color intensity in plant organs is determined by the accumulation of flavonoids, particularly anthocyanins and flavones (Harborne etal., 1990; Mizuno etal., 2015; Nordström and Swain, 1953; Tanaka etal., 2008). Prevailing environmental conditions influence flavonoid accumulation, as in the case of anthocyanin accumulation in Arabidopsis thaliana (Rowan etal., 2009). In many plant species, the accumulation of flavonoids in plant organs decreases under high temperature conditions. In grapes, high night temperatures reduced anthocyanin accumulation in the grape skin the quality of berries (Mori etal., 2005). In the ‘Nessho’ red dahlia cultivar, high summer temperatures result in the production of red flowers whereas low winter temperature results in orange flowers (Okada etal., 2020). Color changing in D.variabilis dark red cultivars results from the accumulation of more flavones and fewer anthocyanins (Deguchi etal., 2013; Thill etal., 2012). Similarly, the accumulation of more flavones instead of anthocyanins led to paler violet Antirrhinum majus flowers (Luo etal., 1991). In our study, the red ray florets of ‘Nessho’ accumulated more pelargonidin and cyanidin-based anthocyanins, while the color-changed orange ray florets accumulated more apigenin and luteolin, both of which are flavones (Fig.2). This finding corresponds with earlier reports on the dahlia ‘Kokucho’, including the original red and its purple mutants (Deguchi etal., 2015). Both anthocyanins and flavones are produced in the general flavonoid pathway, with flavones being a product of the branch pathway from chalcones. A control of gene expression and enzymes encoding their activities are crucial to the balance at branch points (Davies etal., 2003). This is a result of substrate competition by the enzymatic activities at these branch points (Deguchi etal., 2015; Takos etal., 2006; Thill etal., 2012). When DvFNS was silenced in black dahlia cultivars, the competition between anthocyanin synthesis and flavone synthesis was disrupted leading to high accumulation of anthocyanins (Deguchi etal., 2013).

To verify the genes responsible for color change in the dahlia ‘Nessho’ cultivar, relative expression analysis was done for major structural genes of the flavonoid biosynthetic pathway, namely, DvCHS1, DvCHS2, DvCHI, DvANS, DvFNS, DvFLS, DvDFR, DvF3H, and DvF3'H in red and orange ray florets. The expression profiles of all genes after the first main branch point on the biosynthesis pathway (at the naringenin stage) were not significantly different between the red and the orange ray florets except for DvFNS (Fig.3a). This result suggests that DvFNS expression may be a major contributing factor to the red to orange color change of the ‘Nessho’ ray florets. The four-fold change in DvFNS expression levels in orange faded sections compared to red regions with variegated ray florets (Fig.3b) further confirmed the possible role of this gene in the flower color change in ‘Nessho’. Expression levels of anthocyanin structural genes (DvDFR, DvF3H, and DvF3'H) did not vary between the red and orange regions. This concurs with a report in Gerbera hybrida, where lines lacking FNSII expression and FNSII activity accumulated more anthocyanin and no flavones, leading to a notable increase in color intensity (Martens and Mithöfer, 2005). In a similar manner, the majority of black dahlia cultivars accumulate more anthocyanins due to low FNSII activity, as well as FNSII expression (Thill etal., 2012). In Lonicera japonica and L.macranthoides, higher expression levels of FNSII were consistent with more flavone accumulation in flowers and flower buds, respectively (Wu etal., 2016).

Having demonstrated that DvFNS is the likely gene responsible for the color change in ‘Nessho’ ray florets, we tried to evaluate whether the expression change occurred only in the ray florets or also in leaves. The results for five leaf developmental stages (Fig.4a) showed that DvFNS expression was elevated in the young unexpanded leaves as compared to leaves on the 1^st–4^th nodes in RH-sensitive plants. This indicates temporal expression of structural genes in the flavonoid biosynthetic pathway in this cultivar. This concurs with a report by Yuan etal. (2013), in which temporal expression profiles of some putative structural genes in various tissues (leaves, stems, stamen, pistil, and petals) revealed a strong correlation with the formation of the red pigment in tulip petals. Temporal expression of CHS genes in the dahlia bi-color cultivar ‘yuino’ was characterized by Ohno etal. (2011b) and was detected in all petal developmental stages except in the young stage, with lower expression in white flower parts than in red parts.

To verify the relationship between DvFNS expression in young unexpanded leaves and seasonal flower color changes, gene expression profiles were compared in winter and summer. A five-fold higher DvFNS expression was observed in winter as compared to summer and this corresponded to flower color changes in the different seasons (Fig.4b). Additionally, a comparison between DvFNS expression in young unexpanded leaves and the color fading index of the capitulum from August through December showed a similar trend (Fig.5). This may indicate temporal DvFNS expression in young leaf tissues that corresponds with flavone accumulation in flowers, as in the case of apigenin accumulation and FNSII expression in Gentiana triflora ‘Maciry’ leaves (Nakatsuka etal., 2005). The positive correlation between DvFNS expression and the color changing index CFI (Fig.6a) agrees with the reports of Deguchi etal. (2013), Deguchi etal. (2015), and Thill etal. (2012) who found that in purple and red dahlia cultivars a decline in the expression of FNSII resulted in a deeper color in majority of the black dahlia cultivars and vice versa in the other colored cultivars such as white, yellow, orange, pink, and red. The negative correlations between CFI and average weekly temperature (Fig.6b) and DvFNS expression in young unexpanded leaves and average weekly temperature further confirm the role of temperature in the expression of the DvFNS gene, accumulation of flavone and consequently flower color change in the ‘Nessho’ dahlia cultivar. Similarly, Pohlia nutans FNSI possesses hypothermic enzyme characteristics, retaining relatively high enzymatic activity in low-temperature environments in Antarctica. This led to high flavone accumulation in moss under low-temperature conditions (Wang etal., 2020).

Some ‘Nessho’ plants are more sensitive to color fading than others in similar environmental conditions. This is evidenced by the varying color-fading indices at the flowering stage and by the difference in gene expression at the young leaf stage (Fig.7). Other factors may be involved in the difference in the expression of DvFNS in RL- and RH-sensitive cultivars at low-temperatures. In the future, identifying the genetic differences between RL- and RH-sensitive plants that cause different DvFNS expression levels will lead to the development of genetically uniform cultivars and high quality flower production.

Conclusion

In conclusion, our study demonstrates that DvFNS expression in the ‘Nessho’ dahlia cultivar plays an important role in the accumulation of flavones in ray florets. Seasonal temperature changes influence the expression of DvFNS, with low winter temperatures up-regulating the expression and high summer temperatures down-regulating it. This results in the production of faded orange flowers in winter. There is also a positive correlation between DvFNS expression in ray florets, in young unexpanded leaves, and color fading occurrence (Fig.8). This is an important finding that can be used in the selection of non-fading lines at the seedling stage.

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