Expression levels of ethylene biosynthetic genes and senescence-related genes in carnation (Dianthus caryophyllus L.) with ultra-long-life flowers

Scientia Horticulturae 183 (2015) 31–38

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Expression levels of ethylene biosynthetic genes and senescence-related genes in carnation (Dianthus caryophyllus L.) with ultra-long-life flowers Koji Tanase a,∗ , Sawako Otsu b , Shigeru Satoh b , Takashi Onozaki a a b

NARO Institute of Floricultural Science (NIFS), National Agriculture and Food Research Organization (NARO), Tsukuba 305-8519, Japan Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan

a r t i c l e

i n f o

Article history: Received 3 March 2014 Received in revised form 18 November 2014 Accepted 27 November 2014 Keywords: Carnation Ethylene biosynthetic gene Flower life Senescence Senescence-related gene Ultra-long-life flower

a b s t r a c t Carnation (Dianthus caryophyllus L.) line 532-6 has ultra-long flower life (29 days without chemical treatment). We investigated ethylene production and expression of ethylene biosynthetic genes and senescence-related genes in the flowers of 532-6 in comparison with those of a control cultivar, ‘Francesco’ and a cultivar with long flower life, ‘Miracle Rouge’ (MR). ‘Francesco’ flowers, but not those of MR and 532-6, showed typical symptoms of ethylene-dependent senescence. The flowers of MR and 532-6 produced very low levels of ethylene as a result of very low expression levels of two ethylene biosynthetic genes, DcACS1 and DcACO1. The expression of some senescence-related genes (DcbGal, DcGST1 and DcLip) increased in ‘Francesco’ petals by day 6, but remained low in the petals of MR and 532-6 throughout the experiment, whereas there was a small difference between the levels of the DcCP1 transcript in MR and 532-6. The expression of DcCPIn was regulated differently in all three cultivars. In ‘Francesco’ petals, the DcCPIn expression level sharply decreased on day 6 and 7; in MR, the DcCPIn level increased on day 7 and decreased by day 16, whereas in 532-6 it increased on day 3 and then gradually decreased until day 25. The results suggest that the extended flower life of MR and 532-6 in comparison with ‘Francesco’ depends on reduced levels of ethylene production, low levels of ethylene biosynthetic gene expression, and senescence-related gene expression; the difference between MR and 532-6 may be due, in particular, to DcCPIn and DcCP1. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

1. Introduction Carnation (Dianthus caryophyllus L.) flowers are sensitive to ethylene, and their senescence is regulated by ethylene produced autocatalytically several days after anthesis (Woltering and van Doorn, 1988; Woodson and Lawton, 1988). The ethylene biosynthesis pathway is as follows: methionine → S-adenosylmethionine (AdoMet) → 1-aminocyclopropane1-carboxylate (ACC) → ethylene (Kende, 1993). The conversion of AdoMet to ACC is catalyzed by ACC synthase (ACS), and the conversion of ACC to ethylene by ACC oxidase (ACO) (Kende, 1993;

Abbreviations: ACC, 1-aminocyclopropane-1-carboxylate; ACO, ACC oxidase; ACS, ACC synthase; AdoMet, S-adenosylmethionine; bGal, ␤-galactosidase; CP, cysteine proteinases; CPIn, cysteine proteinase inhibitor; GST, glutathione-Stransferase; HQS, 8-hydroxyquinoline sulfate; Lip, lipase; MR, Miracle Rouge; SR, senescence-related; STS, silver thiosulfate anionic complex. ∗ Corresponding author. Tel.: +81 29 838 6801. E-mail address: [email protected] (K. Tanase).

Yang and Hoffman, 1984). During carnation flower senescence, ACS and ACO expression and the autocatalytic ethylene production lead to subsequent petal in-rolling, which is a typical form of wilting. Three genes encoding ACS (DcACS1, previously described as CARACC3, DcACS2 and DcACS3) and one gene encoding ACO (DcACO1, previously described as pSR120) have been identified and their expression patterns examined (Henskens et al., 1994; Jones and Woodson, 1999; Park et al., 1992). DcACO1 and DcACS genes are expressed in gynoecia and petals; DcACS1 is the predominant DcACS gene expressed in petals and DcACS2 and DcACS3 are expressed in gynoecia (Jones and Woodson, 1999; Satoh and Waki, 2006). The expression of DcACS1 and DcACO1 is enhanced by exogenous ethylene, which induces ethylene production. Flowers of some carnation cultivars have a long life, which is associated with low ethylene production and low ethylene sensitivity (Wu et al., 1991). Some cultivars and lines with low ethylene production in flowers are ‘Killer’ (Serrano et al., 1991), ‘Sandra’ (Wu et al., 1991), ‘Sandrosa’ (Mayak and Tirosh, 1993), lines 87-37G2 and 81-2 (Brandt and Woodson, 1992), ‘White Candle’ (Nukui

http://dx.doi.org/10.1016/j.scienta.2014.11.025 0304-4238/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

K. Tanase et al. / Scientia Horticulturae 183 (2015) 31–38

Table 1 Flower life of carnation (Dianthus caryophyllus L.). STS, 2 mM silver thiosulfate for 20 h, followed by distilled water. Zn, 2 mM ZnCl2 throughout the experiment. STS + Zn, STS treatment as above, followed by 2 mM ZnCl2 . Values are means ± SE of nine flowers. Different letters indicate a significant difference with P < 0.05 by Tukey’s test. Cultivar or line

Treatment

Flower life (days)

‘Francesco’ (normal life)

– STS Zn STS + Zn

9.1a ± 1.2 21.2b ± 3.1 9.6a ± 0.8 22.6b ± 1.6

– STS Zn STS + Zn – STS Zn STS + Zn

22.7b ± 0.4 22.5b ± 1.6 24.8b ± 0.9 24.8b ± 3.6 29.0c ± 0.8 29.0c ± 4.1 28.5c ± 1.2 31.3c ± 3.1

‘Miracle Rouge’ (long life)

532-6 (ultra-long life)

A Ethylene production (nl g-1 h-1)

32

Whole flowers 45

Francesco (control)

40 35

Miracle Rouge (long life)

30

532-6 (ultra-long life)

25 20 15 10 5 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Days after harvest

Gynoecium Ethylene production (nl g-1 h-1)

B

60

Francesco (control) 50

Miracle Rouge (long life) 532-6 (ultra-long life)

40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Days after harvest

Fig. 1. Flowers of carnation (Dianthus caryophyllus L.) ‘Miracle Rouge’ and line 532-6 on day 25. The cut flowers were kept in distilled water at 23 ◦ C, 70% relative humidity and 12-h photoperiod under cool-white fluorescent lamps (10 ␮mol m−2 s−1 ).

et al., 2004), ‘Miracle Rouge’ (MR), ‘Miracle Symphony’, and lines 006-13 and 62-2 (Onozaki et al., 2006; Tanase et al., 2008, 2013), which have a life of 10–20 days. The expression of DcACS1, but not DcACO1, is low throughout the flower life in ‘White Candle’ (Nukui et al., 2004). Our previous study showed that by comparing cultivar with normal flower life, the expression of DcACS1, DcACS2 and DcACO1 was suppressed in the flowers of MR and ‘Miracle Symphony’, which resulted in a very low level of ethylene production (Tanase et al., 2008). In particular, low DcACO1 expression seems to be linked to the low ethylene production in both cultivars. Many other factors play a role in the regulation of ethylene biosynthesis and petal wilting in senescing carnation flowers (Hoeberichts et al., 2007; Otsu et al., 2007). Petal wilting may be caused by degradation of cellular components, which induces cell death and is mediated by hydrolytic enzymes and their regulators, such as lipases [Lip (Hong et al., 2000; Kim et al., 1999a)], cysteine proteinases [CPs (Jones et al., 1995)] and a cysteine proteinase (CP) inhibitor [CPIn (Kim et al., 1999b; Sugawara et al., 2002)]. Many senescence-related (SR) genes (including DcLip, DcCP1 and DcCPIn) have been cloned from carnation petals and their expression patterns examined. The transcript levels of genes encoding a CP [DcCP1; previously described as pDCCP1 (Jones et al., 1995)], ␤-galactosidase [DcbGal; previously described as SR12 (Lawton et al., 1989)], glutathione-S-transferase (DcGST1) and lipase (DcLip) are increased during flower senescence and by ethylene

Ethylene production (nl g-1 h-1)

C

Petals 60

Francesco (control) 50

Miracle Rouge (long life) 532-6 (ultra-long life)

40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Days after harvest Fig. 2. Ethylene production in carnation flowers during flower senescence. (A) Whole flower. (B) Gynoecium. (C) Petals. Values are means ± SE of three flowers.

treatment (Hong et al., 2000; Kim et al., 1999a; Verlinden et al., 2002), whereas the DcCPIn transcript level decreases under these condition (Sugawara et al., 2002; Tanase et al., 2013). Recently, an ultra-long-life carnation line, 532-6, was reported with a flower life of over 27 days, which is approximately four times that of normal cultivars (Onozaki et al., 2011) and longer than that of any other long-life cultivars studied previously. To obtain clues as to the mechanisms underlying flower longevity in 532-6, we analyzed the expression profiles of ethylene biosynthetic genes and SR genes during flower senescence. We also assessed the effects of

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Fig. 3. Transcript levels of DcACS1 (A) and DcACO1 (B) in the gynoecium and petals during flower senescence. Values are means ± SE of three flowers. ACS, ACC synthase; ACO, ACC oxidase.

exogenous ethylene on gene expression and endogenous ethylene production. 2. Materials and methods 2.1. Plant material The carnation breeding line 532-6 (ultra-long flower life), cultivar ‘Miracle Rouge’ (long flower life), and the control cultivar ‘Francesco’ were grown under natural-daylight conditions in a greenhouse (Onozaki et al., 2011). Hereafter, both cultivars and the breeding line are referred to as ‘cultivars’. Flowers were harvested when the outer petals were horizontal (the full open stage, defined as day 0).

sulfate (HQS) at 23 ◦ C and 24-h photoperiod under cool-white fluorescent lamps (10 ␮mol m−2 s−1 ). HQS was used in all solutions to prevent the growth of microorganisms in the vase water and xylem vessels during long observation periods (>20 days); it is known to be efficient and safe for many cut flowers including carnations (Hassan and Schmidt, 2004). For Zn treatment, stems were kept in a solution containing 2 mM ZnCl2 throughout the experiment. For silver thiosulfate anionic complex (STS) treatment, stems were kept in a solution containing 2 mM STS for 20 h, and then transferred to a solution without STS. For STS + Zn treatment, flower stems were kept in a solution containing 2 mM STS for 20 h, and then transferred to a solution containing 2 mM ZnCl2 . Flowers were evaluated daily. 2.4. Ethylene treatment

2.2. Determination of flower longevity The flower life was determined as the number of days from day 0 until the day of the loss of ornamental value, which was defined as any one of the following: (1) flower wilting with inrolling, (2) browning of the petal margin without in-rolling or (3) other forms of petal desiccation without in-rolling. Flowers were evaluated daily under standard conditions [23 ◦ C, 70% relative humidity, 12-h photoperiod under cool-white fluorescent lamps (10 ␮mol m−2 s−1 )]. 2.3. Cut flower treatment On day 0, stems of the harvested flowers were trimmed to 30 cm and kept in distilled water containing 0.01% 8-hydroxyquinoline

Flowers on day 0 were placed in a 70-l chamber with 10 ␮l l−1 ethylene at 23 ◦ C for 20 h. Flowers were monitored every hour by using a digital camera Caplio GX (Richo, Tokyo, Japan) to determine the beginning of petal in-rolling as a measure of ethylene sensitivity. 2.5. Ethylene production measurement Flowers were exposed to ambient air for 1 h before incubation. Whole flowers were placed into a 143- or 433-ml glass bottle, which was closed with a silicon cap and kept at 23 ◦ C for 1 or 2 h. Gynoecium and petals were placed into a 15-ml glass vial or a glass bottle, which was closed with a silicone cap and kept at 23 ◦ C for 1 h. Gas samples (1 ml) were taken from the headspace

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K. Tanase et al. / Scientia Horticulturae 183 (2015) 31–38

Fig. 4. Transcript levels of DcCP1 (A), DcGST1 (B), DcCPIn (C), DcLip (D), DcbGal (E) and DcCP2 (F) in petals during flower senescence. Values are means ± SE of three replications. CP, cysteine proteinase; GST, glutathione-S-transferase; CPIn, CP inhibitor; Lip, lipase; bGal, ␤-galactosidase.

and injected into a gas chromatograph (GC-13A, Shimadzu, Kyoto, Japan), equipped with an alumina column and a flame ionization detector. 2.6. Quantitative real-time PCR analysis We determined the expression levels of DcACS1 (accession number, M81882.1), DcACO1 (AB042320.1), DcCP1 (U17135.1), DcCP2 (AB177386.1), DcCPIn (AY028994.1), DcbGal (CF259486.1), DcGST1 (L05915.1) and DcLip (BD268753.1) in carnation flowers by using quantitative real-time PCR (qPCR) analysis as described previously (Tanase et al., 2008, 2013). Total RNA was extracted from gynoecia and petals (on day 0, 3, 6, 7, 10, 16, 20 and 25) by using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized on total RNA (1 ␮g) with an oligo (dT) primer and reverse transcriptase by using the Advantage RT-for-PCR kit (BD Biosciences Clontech, Palo Alto, CA, USA). A fragment of a carnation

actin gene (DcACT1-2) was used as an internal control (Tanase et al., 2008). qPCR was performed with SYBR Premix Ex Taq II polymerase (Takara Bio, Shiga, Japan) on a LightCycler model 3.1 System (Roche Diagnostics, Mannheim, Germany). The products were detected by monitoring the increase in fluorescence of the reporter dye at each PCR cycle. For each gene, cDNA was amplified by RT-PCR, cloned into the pT7 Blue vector (Merck Chemicals, Darmstadt, Germany), sequenced and used in the qPCR standard curve assay.

3. Results 3.1. Flower life and senescence symptoms Non-treated ‘Francesco’, MR and 532-6 flowers differed greatly in longevity, which was approximately 9, 23 and 29 days,

K. Tanase et al. / Scientia Horticulturae 183 (2015) 31–38

A: Ethylene production 120

Ethylene production (nl g -1h-1)

respectively (Table 1). ‘Francesco’ flowers, but not those of MR and 532-6, showed in-rolling at senescence. MR flowers slowly faded and showed browning from the petal edge and petal desiccation, whereas petals of 532-6 did not brown and desiccated much more slowly (Fig. 1). STS treatment significantly extended the life of ‘Francesco’ flowers, but not that of MR and 532-6 flowers. Zn treatment did not extend the flower life of ‘Francesco’ and 532-6, and extended it only slightly and not significantly in MR. In comparison with STS treatment, STS + Zn treatment had no effect on the flower life of ‘Francesco’, and extended it slightly but not significantly in MR. The mean response time of the onset of petal in-rolling after ethylene treatment (10 ␮l l−1 ) was similar for ‘Francesco’ (7.7 ± 0.4 h), MR (7.2 ± 0.4 h) and 532-6 (7.0 ± 0.3 h).

35

*

100

Control

Ethylene treatment

80

*

60

*

40 20 0 Francesco (control)

Miracle Rouge (long life)

532-6 (ultra-long life)

B: DcACS1 3.2. Ethylene production by flowers during senescence

In ‘Francesco’, the DcACS1 transcript levels in the gynoecium and petals increased at days 6 and 7 to much higher levels than those in MR (Fig. 3A). In MR, the DcACS1 transcript level slightly increased by day 16 in the gynoecium, but remained low in petals. In 532-6, the DcACS1 transcript levels in the gynoecium and petals were low throughout the experiment. In ‘Francesco’, the DcACO1 transcript levels increased in the gynoecium and petals at days 6 and 7; they peaked at day 6 in gynoecia but continued to increase until day 7 in petals (Fig. 3B). In MR and 532-6, the DcACO1 transcript levels were low in the gynoecium and undetectable in petals throughout the experiment The DcCP1 transcript was detectable at day 0 in the petals of all cultivars; its levels rapidly increased until day 7 in ‘Francesco’, but remained low in MR and 532-6 (Fig. 4A). There was a small but non-negligible difference between MR and 532-6 (Fig. 4A, inset). The DcGST1 transcript levels increased by days 6 and 7 in the petals of ‘Francesco’, but remained very low in those of MR and 532-6 (Fig. 4B). High levels of the DcCPIn transcript were detected at day 0 in the petals of ‘Francesco’ and MR; DcCPIn levels decreased markedly by days 6 and 7 in ‘Francesco’, but first increased (until days 6 and 7) and then decreased by day 16 in MR (Fig. 4C). In 532-6 petals, the DcCPIn level was lower at day 0 than in ‘Francesco’ and MR, but peaked at day 3 and then gradually decreased until day 25 (Fig. 4C). The DcLip transcript level increased at days 6 and 7 in the petals of ‘Francesco’; it also gradually increased in MR and 532-6 petals, but to levels lower than in ‘Francesco’ (Fig. 4D). The DcbGal transcript levels peaked at day 6 in the petals of ‘Francesco’, but remained low in MR and 532-6 petals throughout the experiment (Fig. 4E). In ‘Francesco’ petals, the DcCP2 transcript level was high at day 0, peaked at day 6 and returned to the initial level at day 7 (Fig. 4F). In MR and 532-6 petals, the DcCP2 transcript levels were similar (and lower than in ‘Francesco’) at day 0. In 532-6, the level of this transcript increased and reached a plateau at days 3–10, and then gradually decreased; in MR, the DcCP2 transcript level decreased by day 3 and remained very low throughout the experiment (Fig. 4F).

Relative gene expression

3.3. Gene expression during senescence

1800

Control

1600

Ethylene treatment

*

1400

*

1200

*

1000 800 600 400 200

0 Francesco (control)

Miracle Rouge (long life)

532-6 (ultra-long life)

C: DcACO1 10

Relative gene expression

In ‘Francesco’, ethylene production by whole flowers peaked at day 6 (Fig. 2A). Ethylene production by the gynoecium increased markedly at day 6 and decreased at day 7 (Fig. 2B), and that by petals increased until day 7 (Fig. 2C). Ethylene production by whole flowers, gynoecium and petals of MR and 532-6 was low throughout the experiment (Fig. 2).

2000

9

Control

8 7

*

6

Ethylene treatment

*

*

5 4 3 2 1 0 Francesco (control)

Miracle Rouge (long life)

532-6 (ultra-long life)

Fig. 5. Ethylene production (A) and the transcript levels of DcACS1 (B) and DcACO1 (C) in petals after exogenous ethylene treatment. Values are means ± SE of three flowers. Statistical significance was calculated by t-test (* P < 0.01 compared to nontreated [control] flower). ACS, ACC synthase; ACO, ACC oxidase.

3.4. Effects of exogenous ethylene on endogenous ethylene production and gene expression In all three cultivars, exogenous ethylene induced endogenous ethylene production at a rate that was slightly higher in ‘Francesco’ petals than in those of MR and 532-6 (Fig. 5A). The DcACS1and DcACO1 transcript levels increased in the ethylene-treated gynoecium (data not shown) and petals (Fig. 5B and C) in all cultivars. The DcCP1, DcbGal, DcGST1, DcLip and DcCP2 transcript levels also increased in the ethylene-treated petals in all cultivars (Fig. 6A, B, D–F). Although the DcCP1 transcript levels slightly increased in ethylene-treated petals of MR and 532-6, they remained lower than in control ‘Francesco’ petals. In contrast to the above five

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K. Tanase et al. / Scientia Horticulturae 183 (2015) 31–38

A: DcCP1

B: DcGST1 30

*

50

Control

40 30 20 10

* Miracle Rouge

5 0

532-6

Francesco

532-6

D: DcLip Control

4.5

Ethylene treatment Relative gene expression

Relative gene expression

Miracle Rouge

5

1200 1000 800 600 400 200

*

0

Francesco

* Miracle Rouge

*

4

Control

Ethylene treatment

3.5 3 2.5 2 1.5

*

1

*

0.5

*

0 Francesco

532-6

Miracle Rouge

532-6

F: DcCP2

E: DcbGal

1000

600 Control

Ethylene treatment

*

Control

800

*

400

900 Relative gene expression

Relative gene expression

*

*

10

1400

300 200

Ethylene treatment

15

*

C: DcCPIn

500

Control

20

0 Francesco

*

25

Ethylene treatment Relative gene expression

Relative gene expression

60

*

100

700 600

Ethylene treatment

*

*

500 400

*

300 200 100 0

0 Francesco

Miracle Rouge

532-6

Francesco

Miracle Rouge

532-6

Fig. 6. Transcript levels of DcCP1 (A), DcGST1 (B), DcCPIn (C), DcLip (D), DcbGal (E) and DcCP2 (F) in petals after exogenous ethylene treatment. Values are means ± SE of three replications. Statistical significance was calculated by t-test (* P < 0.01: compared to control flower). CP, cysteine proteinase; GST, glutathione-S-transferase; CPIn, CP inhibitor; Lip, lipase; bGal, ␤-galactosidase.

transcripts, the DcCPIn transcript level in control petals was higher than that in ethylene-treated petals in all cultivars (Fig. 6C).

4. Discussion To identify the key factor(s) that determine the ultra-long-life flower trait in carnation, we compared the expression of genes involved in ethylene biosynthesis and SR genes in cultivars with normal flower life (‘Francesco’), long flower life (MR) and ultralong flower life (532-6). Genotypic variation of the flower life in

carnation is closely related to variation in the ability to synthesize ethylene during flower senescence and to petal sensitivity to ethylene; these traits seem to be heritable (Onozaki et al., 2001). In cultivars producing low ethylene levels (such as MR, ‘Miracle Symphony’ and ‘White Candle’), long flower life is caused by the reduced expression of ethylene biosynthetic genes (Tanase et al., 2008). In the flowers of MR and ‘Miracle Symphony’, the expression of DcACS1 and especially DcACO1 is suppressed, resulting in extremely low ethylene production, whereas in ‘White Candle’ the low expression of DcACS1, but not DcACO1, correlates with the long flower life (Nukui et al., 2004). We found that DcACS1 and DcACO1

K. Tanase et al. / Scientia Horticulturae 183 (2015) 31–38

expression was suppressed in the 532-6 flowers (Fig. 3), suggesting that similar mechanisms cause low ethylene production in 532-6, MR and ‘Miracle Symphony’. Yet, the difference in the flower life between MR and 532-6 cannot be explained by the difference in ethylene production or expression levels of ethylene biosynthetic genes, because ethylene production and the DcACS1 and DcACO1 transcript levels in the petals of the two cultivars were similar. Another factor affecting carnation flower longevity is ethylene sensitivity (Woltering and van Doorn, 1988; Woodson and Lawton, 1988). The timing of the onset of petal in-rolling after exogenous ethylene treatment was similar in ‘Francesco’, MR and 532-6, suggesting similar ethylene sensitivity. In the petals of all three cultivars, exogenous ethylene treatment triggered autocatalytic ethylene production and induction of DcACS1 and DcACO1 expression (Fig. 5). Therefore, ethylene sensitivity cannot explain the difference in flower longevity between MR and 532-6. In a previous study, we compared SR gene expression levels during flower senescence in carnation cultivars with long flower life (Tanase et al., 2013). There were small differences in ethylene production among these cultivars. Some of them, which had residual ethylene production in petals, also had increased expression of DcCP1, DcGST1, DcLip and DcbGal; such cultivars had a slightly shorter flower life than ‘Miracle Symphony’, which had no residual ethylene production. In our present study, the expression of these genes was induced by exogenous ethylene (Fig. 6A, B, D and E). Analysis of promoter cis-elements detected ethylene-responsive elements (ERE) or ERE-like sequences in the 5 -upstream regions of these genes (Itzhaki et al., 1994; Kosugi et al., 2007; Verlinden et al., 2002); thus, the regulation of DcCP1, DcGST1, DcLip and DcbGal expression in carnation petals is likely to be ethylene-dependent. The pattern of DcCP2 expression observed in the long-life carnations in our present study (Fig. 4F) differs from that described previously by Kosugi et al. (2007), who reported that DcCP2 expression remained steady from the full-open to wilting stages and was not responsive to ethylene or dehydration; the latter data suggested that DcCP2 is not involved in petal wilting, though its role remained unclear. In our present study, the expression of DcCP2 in the 532-6 petals was higher than in the MR petals (Fig. 4F); thus, the expression of DcCP2 may depend on the cultivar. The expression of DcGST1, DcLip and DcbGal in petals during senescence was similar in MR and 532-6 (Fig. 4B–E), whereas the expression of DcCP1 slightly differed (Fig. 4A). Thus, we assume that the difference in the expression of DcCP1 might be related to the difference in flower longevity between MR and 532-6. In contrast to DcGST1, DcLip and DcbGal, the expression of DcCPIn was different in the petals of MR and 532-6 during senescence: it was delayed in the 532-6 petals in comparison with MR petals (Fig. 4C). Exogenous ethylene decreased DcCPIn expression in both cultivars (Fig. 6C); DcCPIn expression is probably ethylenedependent and is regulated by senescence. In Iris, ZnCl2 inhibited endoprotease activity in tepals and delayed visible flower senescence (which occurred on day 4) by approximately 8 h (Pak and van Doorn, 2005). However, as the flower life of MR was already over 20 days, ZnCl2 may be less effective at producing an increase in flower life (Table 1). Thus, other factors not implicated in CP inhibition are likely involved in extending the flower life of MR. Completion of the carnation whole-genome sequence will help us to identify the gene(s) implicated in the ultra-long-life trait. The predicted amino acid sequence of CPIn contains a motif necessary for CP binding, and recombinant CPIn inhibited papain (Kondo et al., 1990) and proteinase activity in carnation petal extracts (Sugawara et al., 2002). According to the MEROPS database (Rawlings et al., 2010; Rawlings and Morton, 2008), DcCPIn belongs to the I25B protease inhibitors, which inhibit the C1A-type plant CP including DcCP1. Thus, DcCPIn may inhibit not only DcCP1 but also other CPs. Plant CP genes are a large family, with over 100

37

annotated sequences in the Arabidopsis genome (García-Lorenzo et al., 2006). In a carnation expressed sequence tag (EST) database, we found 57 ESTs corresponding to potential CP genes (Tanase et al., 2012). The CP activity can be controlled at different levels, such as by transcriptional regulation or post-translational processes, and by specific protease inhibitor proteins, including CPIn (Bode and Huber, 1996; Solomona et al., 1999). These mechanisms regulate diverse CP functions in programmed cell death, organ development and responses to pathogens (Chichkova et al., 2004; Solomona et al., 1999; Zhang et al., 2009). We believe that DcCPIn acts as a suppressor of petal senescence in both the long-life cultivar, MR and the ultra-long-life cultivar, 532-6, and the difference in the decrease in DcCPIn expression may cause the difference in MR and 532-6 flower longevity. Thus, we speculate that the ultra-long-life trait in carnation is controlled by CPIn and possibly CP1. This would not exclude a role of other CPIn(s) encoded in the carnation genome acting as suppressors of petal senescence, or other factors unrelated to CP inhibition. Further studies are needed to distinguish between these possibilities.

Acknowledgments We are grateful to Y. Sase and H. Matsumoto for technical assistance. This work was supported by JSPS KAKENHI Grant number 20580041.

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