Gene Expression Analysis of NAC Transcription Factors Related to Flower Senescence in Dahlia

Abstract

Dahlia (Dahlia variabilis) flowers exhibit wide variety in terms of color, shape, and size; however, their short vase life limits their utility as ornamental plants. This study aimed to identify candidate genes related to flower senescence in dahlia using RNA-seq analysis. In total, 2,698 transcription factors were identified in dahlia flowers, with 114 genes belonging to the NAC family. Additionally, FPKM levels of five NAC transcription factors (DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5) increased from flowering to senescence (petal wilting) in the dahlia ‘Kamakura’. Therefore, the expression level of these NACs was investigated in three dahlia cultivars ‘Kamakura’, ‘Micchan’ and ‘Port Light Pair Beauty’ by real-time PCR. The expression level of all five NACs increased from flowering to flower senescence in the three cultivars. The increase in DvNAC1, DvNAC2, DvNAC3, and DvNAC5 expression was delayed in ‘Micchan’ compared to that in ‘Kamakura’ and ‘Port Light Pair Beauty’. This delay in DvNAC1, DvNAC2, DvNAC3, and DvNAC5 expression corresponded with ‘Micchan’ having a longer vase life than the other two cultivars. Furthermore, ethylene treatment accelerated flower senescence and increased NACs expression, except for DvNAC4, while 1-methylcyclopropene delayed both flower senescence and the increase in NAC expression in the three cultivars. These results indicate that NAC transcription factors, especially DvNAC1, DvNAC2, DvNAC3, and DvNAC5, are likely to be genes associated with flower senescence in dahlia.

Introduction

Cut dahlia (Dahlia variabilis) flowers have recently become popular in Japan because of their variation in terms of color, shape, and size. Despite their popularity, they have a short vase life of 3–7 d at room temperature without preservative treatment, which is shorter than many other floricultural crops, including carnation (Dianthus caryophyllus), chrysanthemum (Chrysanthemum spp.), lily (Lilium spp.), and rose (Rosa spp.), which are all ≥ 10 d (Ichimura et al., 2011). Extending the vase life of cut dahlia requires understanding of the causes of flower senescence.

Ethylene promotes flower senescence phenomena such as petal wilting and abscission. Ethylene production increases during flower senescence in some floricultural crops such as carnation, dendrobium orchid (Dendrobium hybrid), eustoma (Eustoma grandiflorum), petunia (Petunia hybrida), and sweet pea (Lathyrus odoratus) (Ichimura et al., 1998; Mor et al., 1984; Porat et al., 1994; van Doorn and Woltering, 2008; Veen, 1979; Wu et al., 1991a, b). Therefore, the vase life of these floricultural crops can be extended by treatment with ethylene inhibitors such as 1-methylcyclopropene (1-MCP) and silver thiosulfate complex (STS) (Ichimura et al., 1998; Mor et al., 1984; Scariot et al., 2014; Veen, 1979, 1983). However, some cultivars of lilies, tulips (Tulipa spp.), and chrysanthemums have low sensitivity to ethylene, making ethylene inhibitors less effective for extending the vase life (Elgar et al., 1999; Han and Miller, 2003; Sexton et al., 2000; Woltering and van Doorn, 1988). Dahlia cultivars, belonging to the Asteraceae family, similar to chrysanthemum, are sensitive to ethylene, and its production increases during flower senescence depending on the cultivar (Azuma et al., 2020; Shimizu-Yumoto and Ichimura, 2013). Therefore, both exogenous and endogenous ethylene promotes petal abscission or wilting in dahlia, with 1-MCP or STS treatment extending vase life (Azuma et al., 2020). Additionally, Yang et al. (2021) indicated that ethylene induces abscission layer formation at the petal-ovary boundaries of dahlia florets, confirming its role in senescence. However, treatments with ethylene inhibitors such as 1-MCP and STS do not dramatically suppress senescence, suggesting other factors are also involved in dahlia flower senescence.

Water relations impairment, carbohydrate deficiency, and ethylene shorten the vase life of cut flowers of many floricultural crops. For example, bacterial proliferation in the vase solution causes vascular occlusion owing to a decrease in hydraulic conductance of cut stems, shortening flower vase life in flowers such as rose and gerbera (van Doorn and de Witte, 1994; Zagory and Reid, 1986). Water relations impairment also shortens the vase life of cut dahlia flowers, although antibacterial treatments such as Kathon CG (mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one) can marginally extend it (Azuma et al., 2019). Furthermore, sucrose treatment promotes flowering, enhances coloration, and prevents discoloration in dahlia (Azuma et al., 2019). However, these treatments do not dramatically suppress flower senescence. Therefore, other factors potentially contribute to flower longevity and senescence in dahlia. Additionally, Onozaki and Azuma (2022) produced three new dahlia cultivars with longer vase life using conventional cross breeding techniques. These cultivars are all progeny of ‘Micchan’, a dahlia with a naturally long flower vase life. These reports suggest the presence of genes that may regulate flower senescence and determine the flower longevity of dahlia flowers.

Genes that belong to the NAC (NAM, ATF1,2 and CUC2) transcription factor family induce phenomena such as senescence and morphogenesis in plants (Nuruzzaman et al., 2013). ANAC029/AtNAP, ANAC059/ORS1, ANAC092/ORE1, and NAC016 promote leaf senescence in Arabidopsis thaliana (Balazadeh et al., 2011; Guo and Gan, 2006; Kim et al., 2013; Rauf et al., 2013). In Japanese morning glory (Ipomoea nil), EPHEMERAL1 (EPH1), belonging to the NAC transcription factor family, was identified as a key regulator of flower senescence (Shibuya et al., 2014). EPH1 expression increases 12 h after flowering, inducing programed cell death of petals and petal wilting (Shibuya et al., 2014). Furthermore, in transgenic Japanese morning glory with suppressed EPH1 expression via RNAi, petal wilting was delayed compared with wild type plants (Shibuya et al., 2014). CRISPR/Cas9-mediated mutagenesis of EPH1 extended flower longevity of the mutant similar to transgenic plants (Shibuya et al., 2018). Ethylene production does not increase during flower senescence, and ethylene inhibitor treatment does not extend flower longevity in Japanese morning glory (Shibuya et al., 2014). Therefore, the EPH1 expression is induced independently of ethylene signaling (Shibuya et al., 2014). However, exogenous ethylene treatment can still induce EPH1 expression and flower senescence in Japanese morning glory (Shibuya et al., 2014).

Similar NAC transcription factors to EPH1 that induce flower senescence have been identified in other floricultural crops such as Japanese gentian, petunia, lilies, and tulip (Luo et al., 2021; Meng et al., 2022; Takahashi et al., 2022; Trupkin et al., 2019). NAC genes are widely present in land plants (Olsen et al., 2005). Therefore, this study aimed to investigate the NAC transcription factors related to flower senescence in dahlia using RNA-seq analysis. Additionally, we compared NAC expression patterns in dahlia cultivars with different vase lives. Dahlia is a plant that belongs to the Asteraceae family, and the form of flower is a capitulum composed of many florets. In dahlia inflorescences, flowering and petal wilting begin from outer whorl petals (Azuma et al., 2019). Therefore, the expression pattern of NAC transcription factors was analyzed by petal position in this study. The study also investigated whether NAC transcription factors in dahlia are induced by ethylene signaling.

Materials and Methods

RNA-seq analysis

The dahlia ‘Kamakura’ was grown in an open field at the College of Bioresource Sciences, Nihon University, Fujisawa, Japan, from April to November 2018, and used for RNA-seq analysis. Dahlia bulbs were purchased in March from Three A Corporation Co., Ltd., Tokyo, Japan. The outermost petals, fully opened in October 2018, were collected as samples. Samples were prepared at four flower developmental stages: 2 d before full opening (day −2), full opening day (day 0), 1 d after full opening (day 1), and complete petal wilting stage (day 8) (Fig. 1). These samples were collected from intact dahlia flowers, not cut flowers. Total RNA was isolated using Fruit-Mate for RNA purification and RNAiso Plus (TaKaRa Bio Inc., Kusatsu, Japan) and treated with recombinant DNaseI (TaKaRa Bio Inc.). The RNA-seq and data analyses were performed by BGI Genomics (Shenzhen, China). About 35.86 Gb bases in total were generated on the BGISEQ-500 sequencing platform. After read filtering, de novo assembly was performed on clean reads using Trinity (Grabherr et al., 2011). The functional annotation of Unigenes was performed using seven functional databases (NR, NT, GO, KOG, KEGG, SwissProt, and InterPro), and a list of 2,698 Unigenes predicted to be transcription factors was compiled. Further classification by transcription factor family revealed that 114 Unigenes belonged to the NAC transcription factor family. The data sets are available in the DDBJ Sequence Read Archive (DRA) (accession number PRJDB20607).

Fig. 1

Flower developmental stages of intact and cut flowers of ‘Kamakura’. A. Intact flowers of ‘Kamakura’ from 2 d before full opening to 11 d after full opening in an open field under natural conditions. B. Cut flowers of ‘Kamakura’ from the day of full opening to 6 d after full opening at 23°C under a 24-h photoperiod.

According to the results of RNA-seq analysis, five NACs—DvNAC1 (Unigene20684_All), DvNAC2 (Unigene18268_All), DvNAC3 (Unigene18209_All), DvNAC4 (Unigene13431_All), and DvNAC5 (Unigene3330_All)— were selected as candidates, and partial sequence cloning of each NAC sequence was performed. The partial sequence cloning was carried out using a Mighty TA-cloning kit (TaKaRa Bio Inc.), and the sequences were determined by Sanger sequencing.

Plant material for NAC gene expression pattern analysis in different dahlia cultivars

We used three dahlia cultivars: ‘Kamakura’, ‘Micchan’ and ‘Port Light Pair Beauty’. Dahlia bulbs were purchased in March 2022 and 2023 from Three A Corporation Co., Ltd., (Tokyo, Japan). Dahlia plants were grown in an open field of the College of Bioresource Sciences, Nihon University, from April to November 2022 and 2023. The outermost petals without ovaries, fully opened, were collected as samples from 2 d before full opening until petal wilting in October 2022 and 2023. These samples were collected by taking two petals from each of two intact flowers (inflorescences), and mixing the four petals to form a single sample. Petals were collected from the same flower for three consecutive days, and for the next three days, petals were collected from a different flower. This sampling was performed three times. Additionally, removing the petals did not affect the vase life of the intact flower (Table S1).

‘Kamakura’ was also used to analyze NAC gene expression patterns in different petal positions. In this experiment, petal samples were collected in different whorls: the outer petal (outermost whorl), middle petals (fourth whorl from the outermost), and inner petal (eighth whorl from the outermost) at 0 and 5 d after full opening. These samples were collected following the same procedure as described above.

Plant material for NAC genes expression pattern analysis under ethylene or 1-MCP treatments

Cut flowers of the dahlia cultivars ‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’ were used in the experiments. All cut flowers were obtained from growers in Asahi, Chiba Prefecture, Japan, in November 2022 and February 2023. The flowers were harvested when the outermost whorl of petals opened. The cut dahlia flowers were wet-transported to our laboratory, taking approximately 1 d.

Three cut flowers from each of the three cultivars were placed in 70-L acrylic boxes containing air only (control), ethylene, or 1-MCP with ethylene. For the ethylene treatment, ethylene was injected by syringe into the acrylic boxes via a septum at a concentration of 10 μL·L⁻¹. For the 1-MCP with ethylene treatment, EthylBloc (Rohm and Haas Japan, Tokyo, Japan) was added to distilled water to release 1-MCP at a concentration of 2.2 μL·L⁻¹ with 10 μL·L⁻¹ ethylene. Throughout the experimental period, these boxes were kept at 23°C with a 24-h light period using cool-white fluorescent lamps with a photosynthetic photon flux density of 15 μmol·m⁻²·s⁻¹. After 24 h, each box was opened for 5 min to release the ethylene, 1-MCP or air, then reclosed, and ethylene or 1-MCP with ethylene was reintroduced at the same concentration. This procedure was repeated daily until petal wilting or abscission was observed. Petals in the fourth row from the outermost whorl (middle petals) were collected as samples each day after treatment. Ethylene sensitivity was evaluated daily by recording the time elapsed from the start of ethylene treatment until petal wilting in the florets of three whorls or abscissions of > 10 petals, confirmed by gently touching the petals by hand.

The petals for RNA extraction were collected by taking one petal from each of three flowers within the same treatment, and the three petals were mixed to form a single sample. Throughout the experimental period, all petals were collected from the same flower. This sampling was repeated three times. Additionally, removing the petals did not affect the vase life of the cut dahlia flowers (Table S1).

Quantitative real-time PCR

Total RNA was isolated the above samples using NucleoSpin RNA Plant and Fungi (TaKaRa Bio Inc.). cDNA synthesis was performed using the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time; TaKaRa Bio Inc.). PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa Bio Inc.) and Thermal Cycler Dice Real Time System III (TaKaRa Bio Inc.). Thermal cycling conditions were set at 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. NAC primers for real-time PCR were designed using Primer3Plus (https://www.primer3plus.com/) and the primers are listed in Table 1. An actin primer, as a housekeeping gene, was used in a similar manner to Ohno et al. (2018).

Table 1

Primers for real-time PCR expression analysis.

Statistical analyses

Data were analyzed using Tukey–Kramer’s multiple range test. BellCurve for Excel (Social Survey Research Information Co., Ltd.) was used for the analysis.

Results

Investigation of NAC transcription factors in dahlia via RNA-seq analysis

Petal wilting of the outermost petals started 5 d after full opening, and was completed by 8 d in intact ‘Kamakura’ dahlia (Fig. 1). RNA-seq analysis was used to identify candidate NAC genes that could induce flower senescence. Collectively, 2,698 transcription factors were identified in dahlia flowers, with 114 genes belonging to the NAC transcription factor family (Fig. S1; Table S2). Among these NAC, Unigene4864_All and Unigene12662_All had high homology with EPH1, but the expression levels were low, and only slight changes in expression levels were observed until the flower senescence stage (Figs. S2 and S3). After analyzing the FPKM from the RNA-seq data, the FPKM of five NACs—DvNAC1 (Unigene20684_All), DvNAC2 (Unigene18268_All), DvNAC3 (Unigene18209_All), DvNAC4 (Unigene13431_All), and DvNAC5 (Unigene3330_All)—was higher at 1 and 8 d after full opening than at 2 d before full opening and on the full opening day (Fig. S4). Furthermore, cloning and alignment of partial sequences of these NACs were conducted, and they were almost identical to Unigene20684_All, Unigene18268_All, Unigene18209_All, Unigene13431_All and Unigene3330_All, respectively (Figs. S5–S9).

Expression patterns of NAC transcription factors

The timing of petal wilting in dahlia varies depending on the cultivar. Under intact flower conditions, petal wilting of the outermost petal was observed 5–6 d after full opening in ‘Kamakura’ and ‘Port Light Pair Beauty’ and 10 d after full opening in ‘Micchan’ (Table 2). The expression patterns of NACs (DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5) in ‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’ were analyzed using real-time PCR. The expression levels of all five NACs increased from full opening to flower senescence in the three cultivars (Figs. 2–4). In ‘Kamakura’, the expression of DvNAC3, DvNAC4, and DvNAC5 increased 4 d after full opening, and the expression of DvNAC1 and DvNAC2 gradually increased thereafter (Fig. 2). In ‘Port Light Pair Beauty’, the expression of DvNAC2 and DvNAC3 increased 4 d after full opening, and the expression of DvNAC1 and DvNAC5 gradually increased thereafter (Fig. 3). In ‘Micchan’ the expression of DvNAC2, DvNAC3, and DvNAC5 increased 8 d after full opening (Fig. 4). DvNAC1 expression increased gradually increased after full opening (Fig. 4). The timing of increased DvNAC1, DvNAC2, DvNAC3, and DvNAC5 expression in ‘Micchan’ which has a long flower vase life, was delayed compared with that of ‘Kamakura’ and ‘Port Light Pair Beauty’, which have short vase lives (Figs. 2–4).

Table 2

Flower longevity of intact dahlia flowers (‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’).

Fig. 2

Expression patterns of NACs in the flower senescence stage of ‘Kamakura’ dahlia. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 in petals were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated.

Fig. 3

Expression patterns of NACs in the flower senescence stage of ‘Port Light Pair Beauty’ dahlia. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 in petals were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated.

Fig. 4

Expression patterns of NACs in the flower senescence stage of ‘Micchan’ dahlia. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 in petals were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated.

Different NACs expression patterns based on inflorescence petal position

The expression levels of NAC transcription factors were investigated in petal positions of inflorescences in ‘Kamakura’. The expression levels of DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 at day 0 showed no differences across petal positions and remained low (Fig. 5).

The expression levels of DvNAC1, DvNAC4, and DvNAC5 at 5 d after full opening increased in the outer, middle and inner petals (Fig. 5). Additionally, DvNAC2 and DvNAC3 at 5 d after full opening increased in the outer and middle petals (Fig. 5). However, in the inner petals, the expression level of DvNAC2 and DvNAC3 showed almost no difference between 0 and 5 d after full opening (Fig. 5).

Fig. 5

Expression level of NACs in outer, middle, and inner petals of ‘Kamakura’ at 0 and 5 d after full opening. The expression patterns of NAC transcription factors (DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5) in the outer petals (outermost whorl), middle petals (fourth whorl from the outermost), and inner petals (eighth whorl from the outermost) were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed triplicate, and averages with standard errors are indicated.

NAC gene expression patterns under ethylene or 1-MCP treatments

Ethylene treatment significantly shortened the time to petal wilting or abscission, which was observed within 3 d in ‘Micchan’, and ‘Port Light Pair Beauty’ (Table 3). The expression levels of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 increased after 2 d of ethylene treatment in ‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’ (Figs. 6–8). Additionally, 1-MCP treatment delayed petal wilting, with wilting observed in ‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’ at 7, 9, and 7 d, respectively (Table 3). The increase in expression levels of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 under 1-MCP treatment was more gradual than that under air control in ‘Kamakura’ and ‘Port Light Pair Beauty’ (Figs. 6 and 7).

Table 3

Vase life of cut dahlia flowers (‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’) after air, ethylene or ethylene plus 1-MCP treatments.

Fig. 6

Expression patterns of NACs under ethylene or ethylene plus 1-MCP treatments in ‘Kamakura’ dahlia. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated. Values labeled with different letters are significantly different (Tukey’s test, P < 0.05).

Fig. 7

Expression patterns of NACs under ethylene or ethylene plus 1-MCP treatments in ‘Port Light Pair Beauty’. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated. Values labeled with different letters are significantly different (Tukey’s test, P < 0.05).

Fig. 8

Expression patterns of NACs under ethylene or ethylene plus 1-MCP treatments in ‘Micchan’. Expression patterns of NAC transcription factors DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 were analyzed using real-time PCR. The values were normalized using Actin as the housekeeping gene. Experiments were performed in triplicate, and averages with standard errors are indicated. Values labeled with different letters are significantly different (Tukey’s test, P < 0.05).

Discussion

This study aimed to investigate the genes related in flower senescence and longevity in dahlia using RNA-seq analysis. RNA-seq analysis identified 2,698 transcription factors from the petals of ‘Kamakura’, with 114 genes belonging to the NAC family (Fig. S1). The NAC family is one of the largest transcriptional factor families in plants, and NAC family genes are implicated in plant growth, development, stress response, and senescence (Yuan et al., 2019). In floricultural crops such as Japanese morning glory, petunia, Japanese gentian, lily and tulip, NAC genes regulate flower senescence and programmed cell death of petals (Luo et al., 2021; Meng et al., 2022; Shibuya et al., 2014; Takahashi et al., 2022; Trupkin et al., 2019). We hypothesized that among the 114 identified NAC genes there are some NAC genes that control flower senescence and compared their FPKM values across four floral development stages. Five NAC genes, DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5, had higher FPKM values after full opening than before (Fig. S4). This result suggests that the expression of these NAC genes regulating flower senescence increases after full opening, before flower senescence. Similarly, the expression level of EPH1 in Japanese morning glory increased 13 h after flowering (Shibuya et al., 2014). The expression of TgNAP in tulip is low from the flower bud stage to flowering and significantly increases during the early senescence phase (Meng et al., 2022).

Therefore, the expression patterns of DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 were investigated during flower developmental stages in ‘Kamakura’, ‘Micchan’, and ‘Port Light Pair Beauty’ using real-time PCR. Results showed that the expression level of DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 increased after full opening in all three cultivars (Figs. 2–4). However, the increase in expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 in ‘Micchan’ were delayed compared with ‘Kamakura’ and ‘Port Light Pair Beauty’ (Figs. 2–4). The vase life of ‘Micchan’ is longer than that of ‘Kamakura’ and ‘Port Light Pair Beauty’, suggesting that the timing of increased NAC expression affected flower vase life. The expression levels of DvNAC1, DvNAC2, DvNAC3 and DvNAC5 significantly increased on the day immediately before petal wilting in all three cultivars. There may be differences between cultivars in both the required expression levels and the timing of NAC expression associated with the promotion of senescence. The long vase life observed in ‘Micchan’ may be attributable not to lower levels of NAC expression, but rather to a more gradual increase in NAC expression.

Additionally, the expression levels of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 were higher in middle petals than those in inner petals at 5 d after full opening (Fig. 5). The expression of these NACs was induced from outer to inner petals, consistent with the order of petal wilting. These results suggest that NAC transcription factors, DvNAC1, DvNAC2, DvNAC3, and DvNAC5, may be regulators of flower senescence in dahlia, with their expression level or timing possibly contributing to flower longevity. However, the increase in DvNAC4 expression level was minimal in all three cultivars. These results indicate that, although these NAC genes, especially DvNAC1, DvNAC2, DvNAC3, and DvNAC5, are induced by age and contribute to dahlia flower senescence, the roles or signals required for the expression of NAC genes may differ. For example, ANAC019, ANAC055, and ANAC072 in Arabidopsis thaliana are implicated in overlapping stress response roles and similar expression patterns (Hickman et al., 2013; Jensen and Skriver, 2014). However, they have different roles in developmental senescence, with partially differing downstream and upstream genes (Hickman et al., 2013; Jensen and Skriver, 2014). Furthermore, ATAF2 in Arabidopsis thaliana, a regulator of biotic stress responses, upregulates several NAC transcription factors such as ORE1, ORS1, and ANAC046, which promote leaf senescence (Nagahage et al., 2020, 2023; Oda-Yamamizo et al., 2016). NAC016 binds to promoters of AtNAP and ORS1, and promotes leaf senescence in Arabidopsis thaliana (Kim et al., 2013). Therefore, it is possible that DvNAC4 may indirectly contribute to dahlia flower senescence, and DvNAC1, DvNAC2, DvNAC3, DvNAC4, and DvNAC5 in dahlia may have slightly different roles, or the signals for their expression may vary. Furthermore, a BLAST search was performed against the RNA-seq data in this study for genes similar to the ATAF2 gene, and DvNAC5 was identified as the one with the highest homology. This result suggests that DvNAC5 may be a factor that regulates tissue senescence, similar to ATAF2.

EPH1 in Japanese morning glory, a key regulator of flower senescence, is expressed in an age-dependent manner; however, its expression is also induced by exogenous ethylene, which accelerates flower senescence (Shibuya et al., 2014). In our study, the expression levels of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 increased following exogenous ethylene treatment, which also promoted flower senescence in cut dahlia flowers (Table 3; Figs. 6–8). However, the expression of DvNAC4 in ‘Micchan’ was not induced by ethylene treatment (Figs. 6–8). These results indicated that the expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 is induced by exogenous ethylene. Dahlia flowers are sensitive to ethylene, and ethylene production during flower senescence varies by cultivar (Azuma et al., 2020; Shimizu-Yumoto and Ichimura, 2013). In ‘Port Light Pair Beauty’, endogenous levels of ethylene production in petals increase 2–3 d after harvest, with petal wilting observed at 4–5 d (Azuma et al., 2020). In our study, the expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 in ‘Port Light Pair Beauty’ increased 3 d after air treatment (Fig. 7), corresponding with the timing of ethylene production. Additionally, ethylene treatment with 1-MCP delayed petal wilting and increasing the expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 transcription factors (Table 3; Figs. 6–8), indicating that endogenous ethylene promotes the expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5. However, 1-MCP treatment with ethylene treatment did not dramatically prevent flower senescence or NAC expression, suggesting that NAC transcription factors are regulated by both ethylene- and age-dependent pathways, similar to EPH1 in Japanese morning glory (Fig. 9; Shibuya et al., 2014). Therefore, ethylene likely induces senescence in many dahlia cultivars, with those producing higher levels of ethylene having shorter vase lives. Additionally, although 1-MCP inhibits the expression of NAC transcription factors and extends vase life, it does not dramatically prevent age-dependent NAC expression and subsequent senescence.

Fig. 9

Model of the expression pathway of NAC transcription factors in dahlia. The NAC transcription factors of dahlia flowers have two expression pathways. The expressions of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 were strongly induced, and the expression of DvNAC4 was gradually induced with time. Additionally, endogenous and exogenous ethylene induced the expression of DvNAC1, DvNAC2, DvNAC3, and DvNAC5.

Conclusions

The flower vase life of dahlia varies depending on the cultivar. In this study, five NAC transcription factors were identified. The expression levels of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 significantly increased before petal wilting. These NAC transcription factors contribute to flower longevity, as their expression was delayed in dahlias with longer vase life. Additionally, the expressions of DvNAC1, DvNAC2, DvNAC3, and DvNAC5 were induced by ethylene, so it was clarified that NAC transcription factors in dahlia have at least two expression pathways: one ethylene-dependent and one age-dependent. Among five NAC transcription factors, DvNAC1, DvNAC2, DvNAC3, and DvNAC5 are potentially key factors promoting flower senescence in dahlia. Suppressing the expression of these NAC transcription factors could potentially extend the flower longevity of dahlia.

Acknowledgements

We thank H. Shiina for preparing cut dahlia flowers. We appreciate technical assistance from N. Kawaguchi, T. Kumasaka, M. Matsuno, K. Yamada., K. Suzuki, and A. Kiyokawa.

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