Regulation of Ethylene- and Senescence-related Genes in Pot Carnation Flowers during Flower Senescence

Abstract

We investigated differences in flower longevity, ethylene production, and ethylene sensitivity among pot carnation cultivars by quantitative PCR analysis. The flower life of ‘Polaris’ was significantly longer than that of ‘Ariel’ (control cultivar), ‘Orange Duo’, and ‘Lemon Soft’. The flowers of ‘Polaris’ produced little ethylene, and had low expression of the ethylene biosynthesis genes DcACS1 and DcACO1. The transcript levels of senescence-related (SR) genes DcCP1, DcbGal, and DcGST1 increased in petals of ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ at senescence, but were very low in ‘Polaris’. These results suggest that the low ethylene production in ‘Polaris’ is caused by low expression of DcACS1 and DcACO1, as in long-life flowers of the cut carnation cultivars ‘Miracle Rouge’ and ‘Miracle Symphony’. The ethylene sensitivity of ‘Orange Duo’ and ‘Lemon Soft’ was lower than that of both ‘Ariel’ and ‘Polaris’. Petals of ‘Orange Duo’ and ‘Lemon Soft’ wilted, and inrolled more slowly than those of ‘Ariel’ and ‘Polaris’, despite the upregulation of ethylene biosynthesis genes (DcACS1 and DcACO1), DcCP1, DcbGal, DcGST1, and DcEIL3 in petals of all cultivars upon application of exogenous ethylene. These results imply that only the components related to the inrolling of petals are altered in these ethylene-insensitive cultivars.

Introduction

In Japan, a traditional present for Mother’s Day began in the 1960s, and cut carnations and pot carnations (potted carnations) are popular Mother’s Day gifts. Almost all of pot carnations are sold on Mother’s Day, so the peak shipping time is just before that day in Japan (Komagata and Motozu, 2010; Komagata et al., 2005). Thus, millions of pot carnations must be shipped within a short period of time. An estimated 5.2 million pot carnations were produced in 2008 (data from Japanese Ministry of Agriculture, Forestry and Fisheries), and 8.2 million were produced in 2011 (data from Japan Flower Promotion Center Foundation: http://jfpc.or.jp/).

During transportation and storage, pot carnations sometime deteriorate because of environmental factors such as low light intensity, temperature fluctuations, and air pollution (Leonard et al., 1995; Yamane et al., 2008, 2010). The flowers are particularly sensitive to these factors and show much more deterioration than the leaves. Ethylene production is the major factor, affecting post-harvest quality of many potted plants that require transportation, including carnations (Woltering and van Doorn, 1988; Yamane et al., 2007). Several methods can be used to prolong flower life, one of which is treatment with post-harvest chemicals. Yamane et al. (2007) suggested that ethylene inhibitors, such as silver thiosulfate and 1-methylcyclopropene, promote flowering and prolong flower longevity of pot carnations. These chemicals are effective for extending flower life, but purchasing and applying them adds to the growers’ production costs.

An alternative to chemical treatment for prolonging flower life is breeding for long vase life (Onozaki et al., 2015). A breeding research program was started at the NARO Institute of Floricultural Science (NIFS) in 1992 to improve the vase life of cut carnation flowers by conventional cross-breeding techniques. This program produced two cultivars with long flower life, ‘Miracle Rouge’ and ‘Miracle Symphony’, with a vase life of 17.7 to 20.7 days under standard conditions (Onozaki et al., 2006). In both cultivars, the expression of the ethylene biosynthesis genes DcACS1, DcACS2, and DcACO1 was suppressed in the gynoecium and petals, resulting in the production of very little ethylene during flower senescence (Tanase et al., 2008). This downregulation of ethylene biosynthesis delayed upregulation of the senescence-related (SR) genes DcCP1, DcbGal, and DcGST1, which promote senescence, in the petals of long-life carnations (Tanase et al., 2013).

Generally, flowers of carnations are highly sensitive to exogenous ethylene (Woltering and van Doorn, 1988), but the long-flower-life cultivars ‘Chinera’ and ‘Epomeo’ have low sensitivity (Wu et al., 1991). Such low-ethylene-sensitive cultivars can resist the effects of exogenous ethylene that may occur during storage and transportation.

In the model plant Arabidopsis, genetic studies have defined the key elements that mediate the response to ethylene (Wang and Ecker, 2002). Ethylene is perceived by members of the ethylene receptor (ER) family, and the ethylene signal starts with the ERs. In carnations, the ER genes DcETR1, DcERS1, and DcERS2 have been cloned, but transcripts of DcERS1 were not detected in flowers (Nagata et al., 2000; Shibuya et al., 2002). Besides ERs, many ethylene signal components, such as EIN3 and EIN3-like (EIL) proteins, have been studied in many plant species. These proteins are nuclear-localized transcription factors that seem to play important roles in the regulation of genes with an ethylene-responsive element in the promoter region (Solano et al., 1998). In carnations, three EILs (DcEIL1/2, DcEIL3, and DcEIL4) are possible regulators of SR genes. In particular, DcEIL3 may play an essential role during flower senescence through the regulation of SR genes (Iordachescu and Verlinden, 2005).

Pot carnations have wide genetic diversity in terms of flower longevity and ethylene sensitivity (Onozaki et al., 2009). As for flower longevity, it varies from 3.6 to 12.1 days, and the response time also varies with the application of exogenous ethylene from about 5.1 to 15.2 h. Some of these differences occur among cultivars of potted carnations, so these variations can be useful for breeding long-life pot carnations, but genetic control of this variations is still unknown. Here, we investigated the flower longevity, ethylene production, and ethylene sensitivity of several pot carnation cultivars. In addition, we analyzed and compared the expression profiles of ethylene biosynthesis genes, SR genes, ER genes, and EIL genes during flower senescence in these cultivars.

Materials and Methods

Plant materials

In a previous study, we identified pot carnation (Dianthus caryophyllus L.) cultivars with low ethylene production or low sensitivity (Onozaki et al., 2009). ‘Ariel’ was selected as a control cultivar with typical normal flower longevity and ethylene sensitivity. ‘Orange Duo’ and ‘Lemon Soft’ were selected for their low ethylene sensitivity, and ‘Polaris’ for its long flower life. These cultivars were grown according to the methods in the previous study (Onozaki et al., 2009) in the greenhouse at NIFS in 2009, 2010, and 2011. Flower harvest started at the beginning of March, and finished in the middle of May. Flowers were harvested when they began to open (when the outer petals were held at right angles to the stem, defined as day 0). They were trimmed to 5 cm, and kept individually in glass vials containing distilled water in an inspection room kept at 23°C and 70% relative humidity with a 12-h photoperiod under cool-white fluorescent lamps (10 μmol·m⁻²·s⁻¹).

Determination of flower longevity

The flower longevity of each cultivar was determined as the number of days from day 0 until the flowers lost their ornamental value, which was defined as flower wilting with inrolling, browning of the petal edge without inrolling, or desiccating without inrolling. Flowers (n = 12) were evaluated daily.

Evaluation of ethylene sensitivity

Flowers (n = 9) harvested on day 0 were placed in a 50-L chamber with 10 μL·L⁻¹ ethylene (10 ppm) at 23°C. Flower images were recorded every hour by a digital camera (Caplio GX; Ricoh, Tokyo, Japan) to determine the response time, which was defined as the time to the start of petal inrolling.

Quantitative real-time PCR analysis of ethylene-treated flowers

Flowers (n = 3) harvested on day 0 were held in a 70-L chamber with 10 μL·L⁻¹ ethylene for 12 h. After treatment, flowers were exposed to ambient air for 1 h, and then gynoecia and petals were detached, and stored at −80°C for RNA extraction.

Measurement of ethylene production

For measurement of ethylene production from the whole flower, flowers were sampled on days 0, 3, 5, 6, and 11. For measurement of ethylene production from the gynoecium and petals, these organs were sampled on day 0, the day before the ethylene peak in whole flowers, and the peak day of ethylene production in whole flowers. Flowers (n = 3) were exposed to ambient air for 1 h before incubation. Whole flowers were placed in a 143-mL glass bottle, or the gynoecium, and petals were placed in a 15-mL glass vial. The vessels were closed with a silicone cap and held at 23°C for 2 h. A 1-mL sample of gas was taken from the headspace, and injected into a gas chromatograph (GC-13B; Shimadzu, Kyoto, Japan) equipped with an alumina column and a flame ionization detector to determine the ethylene concentration. The carrier gas flow rate was 40 mL·m⁻¹, and the temperatures of the oven, injector, and detector were 80°C, 100°C, and 200°C, respectively.

Quantitative real-time PCR analysis

We used quantitative real-time PCR (qPCR) analysis to determine the expression of ethylene biosynthesis genes (DcACS1, DcACO1), SR genes (DcCP1, DcbGal, DcGST1), a DcCP1 inhibitor gene (DcCPIn), ER genes (DcETR1, DcERS1, DcERS2), and EIL genes (DcEIL1/2, DcEIL3, DcEIL4) in carnation flowers as described previously (Tanase et al., 2008, 2011, 2013). Gynoecium and petals were sampled on days 0, 3, and 5 in ‘Ariel’ and ‘Orange Duo’, on days 0, 3, and 6 in ‘Lemon Soft’, and on days 0, 3, 6, and 11 in ‘Polaris’. Total RNA was extracted from the gynoecium and petals by using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 μg total RNA with an oligo (dT) primer and reverse transcriptase in an Advantage RT-for-PCR kit (BD Bioscience Clontech, Palo Alto, CA, USA). A fragment of a carnation actin gene (DcACT1-2) was used as an internal control (Tanase et al., 2008). For the qPCR standard curve assay, cDNA of each gene was amplified by RT-PCR, cloned into the pT7Blue vector (Merck Chemicals, Darmstadt, Germany), and sequenced. RT-qPCR was performed on a LightCycler model 3.1 system (Roche Diagnostics, Mannheim, Germany).

Results

Flower life, ethylene sensitivity of flowers, and ethylene production by flowers

Flower life of pot carnations differed greatly among the cultivars. The flower life of ‘Polaris’ was significantly longer than ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ (Table 1). At the time of senescence, flowers of ‘Polaris’ showed browning from the petal margins but no inrolling, while those of the other three cultivars showed inrolling.

Table 1

Flower life of pot carnation cultivars.

The ethylene sensitivity of the flowers also differed among the cultivars. ‘Orange Duo’ and ‘Lemon Soft’ showed a significantly more delayed response to exogenous ethylene than ‘Ariel’ and ‘Polaris’ (Table 2). All cultivars eventually showed inrolling as a senescence symptom. Thus, ‘Orange Duo’ and ‘Lemon Soft’ flowers were considered to have low ethylene sensitivity.

Table 2

Response time to exogenous ethylene treatment.

Ethylene production by whole flowers, gynoecium, and petals of ‘Ariel’ and ‘Orange Duo’ increased on day 5 with senescence, and that by ‘Lemon Soft’ increased on day 6 (Fig. 1). However, ethylene production by ‘Polaris’ remained very low throughout the experiment. Ethylene production by gynoecium and petals of ‘Ariel’ and ‘Orange Duo’ increased on day 5, and that by ‘Lemon Soft’ increased on day 6 (Fig. 2A, B). Ethylene production by gynoecium and petals of ‘Polaris’ slightly increased on day 6, but was low compared with that of ‘Ariel’ on day 5.

Fig. 1

Ethylene production in pot carnation flowers during flower senescence. Each value is the mean ± SE of 3 flowers.

Fig. 2

Ethylene production and transcript levels of DcACS1 and DcACO1 in gynoecium and petals during flower senescence. (A) Ethylene production in gynoecium. (B) Ethylene production in petals. (C) DcACS1 in gynoecium. (D) DcACS1 in petals. (E) DcACO1 in gynoecium. (F) DcACO1 in petals. Each value is the mean ± SE of 3 replications.

Expression analysis of ethylene biosynthesis, SR, ER, and EIL genes

The transcript levels of ethylene biosynthesis genes DcACS1 and DcACO1 in the gynoecium of ‘Orange Duo’, ‘Ariel’, and ‘Lemon Soft’ increased on days 3, 5, and 6, respectively, but those in ‘Polaris’ remained low throughout the experiment (Fig. 2C, E). The trends were similar for DcACS1 and DcACO1 in petals (Fig. 2D, F). The transcript levels of both genes increased on day 5 in ‘Ariel’ and ‘Orange Duo’ and on day 6 in ‘Lemon Soft’, whereas those in ‘Polaris’ remained low throughout the experiment.

Transcript levels of the SR genes DcCP1, DcbGal, and DcGST1 in petals of ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ increased on day 5 or 6, but those in ‘Polaris’ remained very low throughout the experiment (Fig. 3A, C, D). Transcript levels of the CP inhibitor gene DcCPIn in ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ peaked or were high on day 3 and decreased quickly on day 5 or 6 (Fig. 3B), but those in ‘Polaris’ decreased slowly from day 3 until the end of the experiment.

Fig. 3

Transcript levels of (A) DcCP1, (B) DcCPIn, (C) DcGST1, and (D) DcbGal in petals during flower senescence. Each value is the mean ± SE of 3 replications.

To identify the relationship between ethylene perception and flower longevity, we determined the transcript levels of ER genes DcETR1 and DcERS2 and EIL genes DcEIL1/2, DcEIL3, and DcEIL4. Transcript levels of DcETR1 in petals of ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ increased on day 5 or 6, but those in ‘Polaris’ increased only slightly on day 6 and decreased again on day 11 (Fig. 4A). Transcript levels of DcERS2 in ‘Ariel’ decrease on day 3, but those in ‘Orange Duo’, ‘Lemon Soft’, and ‘Polaris’ increased on day 3, and decreased subsequently (Fig. 4B). Transcript levels of DcEIL1/2 and DcEIL3 in ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ increased on day 5 or 6 with senescence, but those in ‘Polaris’ decreased slowly from day 6 until the end of the experiment (Fig. 4C, D). Transcript levels of DcEIL4 remained constitutively low in ‘Orange Duo’, ‘Lemon Soft’, and ‘Polaris’ but gradually increased in ‘Ariel’ (Fig. 4E).

Fig. 4

Transcript levels of (A) DcETR1, (B) DcERS2, (C) DcEIL1/2, (D) DcEIL3, and (E) DcEIL4 in petals during flower senescence. Each value is the mean ± SE of 3 replications.

Effects of exogenous ethylene treatment on transcripts of ethylene biosynthesis, SR, ER, and EIL genes

Flowers harvested on day 0 were treated with exogenous ethylene, and ethylene production and transcripts of ethylene biosynthesis genes, SR genes, ER genes, and EIL genes in petals were measured. Exogenous ethylene treatment induced ethylene production in all cultivars (Fig. 5A). Transcript levels of ethylene biosynthesis genes DcACS1 and DcACO1 and SR genes DcCP1, DcGST1, and DcbGal were considerably higher in the ethylene-treated petals than in the control in all cultivars (Fig. 5B, C, D, F, G). In contrast, transcript levels of the DcCP1 inhibitor DcCPIn were lower in the ethylene-treated petals than in the control in all cultivars (Fig. 5E). Transcript levels of the ER genes DcETR1 and DcERS2 were only slightly altered by ethylene treatment in all cultivars (Fig. 6A, B). Transcript levels of the EIL gene DcEIL1/2 decreased by ethylene treatment, but those of DcEIL3 increased, in all cultivars (Fig. 6C, D). Transcript levels of DcEIL4 were unaffected by ethylene treatment in ‘Orange Duo’, ‘Lemon Soft’, and ‘Polaris’, but were increased in ‘Ariel’ (Fig. 6E).

Fig. 5

(A) Ethylene production and (B–G) transcript levels of (B) DcACS1, (C) DcACO1, (D) DcCP1, (E) DcCPIn, (F) DcGST1, and (G) DcbGal in petals after exogenous ethylene treatment. Each value is the mean ± SE of 3 flowers.

Fig. 6

Transcript levels of (A) DcETR1, (B) DcERS2, (C) DcEIL1/2, (D) DcEIL3, and (E) DcEIL4 in petals after exogenous ethylene treatment. Each value is the mean ± SE of 3 replications.

Discussion

Several previous studies have shown that cultivars of cut carnations with low ethylene production reduce ethylene production by different mechanisms. For example, the expression of ethylene biosynthesis genes DcACS1 and DcACO1 was reduced in the flowers of the long-flower-life cultivars ‘Miracle Rouge’ and ‘Miracle Symphony’, whereas expression of DcACS1 (but not DcACO1) was reduced in the flowers of ‘White Candle’ (Nukui et al., 2004; Tanase et al., 2008). Expression of DcACS1 and DcACO1 is also low in other long-life breeding cultivars and lines, including line 532-6, which is the progeny of full-sib lines of ‘Miracle Rouge’ (Tanase et al., 2013, 2015). In the present study, we compared flower longevity, and examined both ethylene production and expression of DcACS1 and DcACO1 in flowers of several pot carnation cultivars. ‘Polaris’ had the longest flower longevity among these cultivars, and it produced very little ethylene (Table 1; Fig. 1). The reduced expression of DcACS1 and DcACO1 in both the gynoecium and petals of ‘Polaris’ (Fig. 2C, D, E, F) suggests a mechanism similar to that in ‘Miracle Rouge’ and ‘Miracle Symphony’. These results indicate that ‘Polaris’ will be useful for breeding long-life pot carnation flowers.

Another factor affecting flower longevity in carnation is sensitivity to exogenous ethylene. The response time (beginning of petal inrolling) after ethylene treatment was similar in ‘Ariel’ and ‘Polaris’, suggesting normal ethylene sensitivity. In both cultivars, ethylene treatment triggered autocatalytic ethylene production, and upregulated ethylene synthesis genes DcACS1 and DcACO1 and SR genes DcCP1, DcGST1, and DcbGal (Fig. 5). Thus, the ethylene sensitivity level of ‘Polaris’ does not influence its long flower life.

In contrast to ‘Polaris’, cultivars with low ethylene sensitivity—‘Orange Duo’ and ‘Lemon Soft’—had comparable flower longevity to the control cultivar, ‘Ariel’, in the absence of exogenous ethylene (Table 1). The mean time to the beginning of petal inrolling after ethylene treatment was 16.1 h in ‘Orange Duo’ and 12.7 h in ‘Lemon Soft’ (Table 2), within the range of ‘Chinera’ and ‘Candy’ under the same ethylene treatment (Onozaki et al., 2004). Further studies will be needed to explain why the flower life of ‘Orange Duo’ and ‘Lemon Soft’ is not extended, whereas other cultivars with low ethylene sensitivity, such as ‘Chinera’, have extended flower life.

In all four cultivars studied here, ethylene treatment induced autocatalytic ethylene production and expression of ethylene biosynthesis genes (DcACS1 and DcACO1) and SR genes (DcCP1, DcGST1, and DcbGal) in petals, while DcCPIn was downregulated (Fig. 5). The DcCP1, DcGST1, and DcbGal which lead to cell death during senescence of petals, were previously thought to be related to the induction of inrolling (Hong et al., 2000; Jones et al., 1995; Kosugi et al., 2000). However, recent research has shown that they are upregulated during senescence regardless of whether inrolling is observed (Otsu et al., 2007; Tanase et al., 2013). Exogenous ethylene treatment for 12 h upregulated these genes in the cultivars with low ethylene sensitivity (Fig. 5), but without inrolling (Table 2). Thus, DcCP1, DcGST1, and DcbGal did not induce petal inrolling directly.

Expression of DcCPIn decreased rapidly in petals of ‘Ariel’, ‘Orange Duo’, and ‘Lemon Soft’ after day 3, and more slowly in petals of ‘Polaris’, which produced less ethylene than the other cultivars during senescence (Figs. 2B and 3B). Its expression was downregulated by exogenous ethylene in all cultivars (Fig. 5E). The level of DcCPIn expression in petals of ‘Polaris’ was similar to that of ‘Ariel’ and ‘Lemon Soft’ on days 5 and 6, even though the flowers of ‘Polaris’ did not exhibit inrolling (Table 1; Fig. 3B). Together, these data show that expression of DcCPIn is ethylene-dependent, and is regulated by senescence, but is unrelated to the ethylene sensitivity of the cultivar. In a previous study, we found that expression of DcCPIn was related to the ultra-long-life trait in cut carnations (Tanase et al., 2015). Further research is needed to elucidate the regulation of DcCPIn expression and its relationship to flower longevity.

In this study, we compared the expression of ethylene signal component genes DcETR1, DcERS2, DcEIL1/2, DcEIL3, and DcEIL4 to clarify the relationship between ethylene signal components and low ethylene sensitivity. ER proteins are considered to act as negative regulators of ethylene response in Arabidopsis (Hua and Meyerowitz, 1998). Loss-of-function mutation in ER genes resulted in strong constitutive ethylene responses. In the tomato, suppressing the ER gene LeETR4 resulted in constitutive ethylene response in transgenic plants (Tieman et al., 2000), and overexpression of another ER gene, Nr, produced an ethylene-insensitive phenotype (Ciardi et al., 2000). Thus, there may be an inverse relationship between ER gene expression and ethylene sensitivity. We speculated that high expression of ER genes would explain the low ethylene sensitivity in flowers of ‘Orange Duo’ and ‘Lemon Soft’. In fact, although the levels of DcETR1 transcripts in these cultivars were 1.5 to 2 times those of ‘Ariel’ and ‘Polaris’ on day 0 and were also higher at most later time points, the levels of DcERS2 transcripts were similar among the four cultivars (Fig. 4A, B). After exogenous ethylene treatment, the expression of DcETR1, DcERS2, DcEIL1/2, and DcEIL4 showed different patterns from those of SR genes (Figs. 5 and 6). Although the relationship between transcript levels of DcETR1 and DcERS2 and ethylene sensitivity in petals remains unclear, DcETR1 and DcERS2 functioned normally as ERs for ethylene perception because exogenous ethylene induced autocatalytic ethylene production in petals (Fig. 5A). In addition, the relationship between DcEIL1/2 and DcEIL4 transcript levels and ethylene sensitivity remains unclear because EIN3/EILs are regulated not only at the mRNA level, but also at the protein level (Yanagisawa et al., 2003).

Among the ER and EIL genes examined, only DcEIL3 was upregulated by exogenous ethylene. Thus, the response of DcEIL3 parallels that of DcACS1, DcACO1, DcCP1, DcGST1, and DcbGal to ethylene (Fig. 6). EIN3, like other EIL proteins, binds to the ethylene-responsive element (ERE) or ERE-like sequences in the 5'-upstream promoter regions of some SR genes such as DcCP1, DcGST1, and DcbGal (Itzhaki et al., 1994; Kosugi et al., 2007; Solano et al., 1998; Verlinden et al., 2002). DcEIL3, which is regulated by ethylene and sugar treatment, may regulate the expression of DcGST1 (SR8) and DcbGal (SR12) (Iordachescu and Verlinden, 2005). In transgenic tomato fruit in which expression of LeEIL was suppressed, ethylene treatment inhibited the induction of LeACS2 and LeACS4 (Yokotani et al., 2009). These results suggest that DcEIL3 plays a crucial role in the petal senescence of pot carnations via the regulation of ethylene biosynthesis genes and of SR genes DcCP1, DcGST1, and DcbGal.

The results reported here imply that cultivars with low ethylene sensitivity—‘Orange Duo’ and ‘Lemon Soft’—possess normal ERs and DcEIL3 function. These cultivars also appear to possess normally functioning components involved in autocatalytic ethylene production such as the products of DcACS1 and DcACO1 and SR genes. Thus, only the signaling components involved in inrolling of petals appear to be altered in these ethylene-insensitive cultivars. We plan to further research the regulation of the components involved in inrolling to elucidate the mechanisms underlying low ethylene sensitivity.

Acknowledgements

The authors are grateful to Dr. S. Satoh, Faculty of Agriculture, Ryukoku University, for his advice. The authors also thank to Mrs. Y. Sase and Mrs. H. Matsumoto at NIFS for technical assistance.

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