Effects of spermine on ethylene biosynthesis in cut carnation (Dianthus caryophyllus L) flowers during senescence

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Effects of Spermine on Ethylene Biosynthesis in Cut Carnation (Dianthus caryophyl/us L.) Flowers During Senescence MYEONG MIN LEE, SUN HI LEE,

and Ky YOUNG

PARK*

Department of Biology, Yonsei University, Sinchon, Seoul 120-749, Korea (M.M.L., S.H.L.); Department of Biology, Sunchon National University, Sunchon, Chonnam 540-742, Korea (K.Y.P.) Received August 1, 1996 . Accepted December 10, 1996

Summary

To investigate the relationship between polyamine and ethylene during senescence of cut carnation

(Dianthus caryophyllus L.) flowers, we studied the effects of spermine on ethylene biosynthesis. Spermine

delayed the senescence of cut carnation flowers and reduced ethylene production, endogenous l-aminocyclopropane-l-carboxylic acid (ACe) content, and the activities and transcript amounts of ACC synthase and ACC oxidase in petals. Methylglyoxal bis-(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, elevated ethylene production, increased activities and amounts of transcripts for ACC synthase and ACC oxidase, and shifted the climacteric pattern of ethylene production ahead by 1 day. However, endogenous ACe content was not increased in the petals of MGBG-treated flowers because of the high activity of ACC oxidase. Spermine also inhibited MGBG-induced ethylene production by decreasing the activities and amounts of transcripts for ACC synthase and ACC oxidase. The accumulation of transcripts for ACC synthase and ACC oxidase in MGBG-treated and in climacteric control petals was correlated with the increase of these enzyme activities. By comparing ethylene production with the changes of endogenous polyamine levels from control and MGBG- or spermine-treated petals during the entire incubation period, it was suggested that endogenous polyamines possibly suppress ethylene production.

Key words: ACC synthase, ACC oxidase, carnation flower, ethylene, MGBG, polyamine, spermine. Abbreviations: ACC = l-aminocyclopropane-l-carboxylic acid; OJ = deionized water; gFw = gram fresh weight; MGBG = methylglyoxal bis-(guanylhydrazone); SAM = S-adenosylmethionine; SAMOC = S-adenosylmethionine decarboxylase. Introduction

Ethylene, a plant hormone, regulates many aspects of plant growth and development (Yang and Hoffman, 1984), and its own biosynthetic enzyme activity with positive or negative mode (Schierle et al., 1989). In carnation flowers the onset of senescence is associated with a sharp increase in ethylene production similar to the climacteric of many fruits (Yang and Hoffinan, 1984). Ethylene biosynthesis is regulated by an autocatalytic mode (Park et al., 1992). * Correspondence.

J Plant Physiol. Vol. 151. pp. 68-73 (1997)

Polyamines and ethylene share a common precursor, Sadenosylmethionine (SAM), but the physiological effects of polyamines and ethylene on senescence and fruit ripening are the opposite of each other (Fuhrer et al., 1982; Winer and Apelbaum, 1986). Biosynthesis of polyamine was inhibited by ethylene in pea seedlings (lcekson et al., 1985), while it was increased by ethylene and auxin in tobacco suspension cells (Park and Lee, 1994). The fact that the ale tomato genotype delays ripening and enhances long keeping properties has been correlated with an increase in the level of putrescine. The cellular contents of polyamines were high in active growing cells, but low in senescing cells concomitant with an

69

Effects of Spermine on Ethylene Biosynthesis

increase in ethylene production in tobacco suspension cultured cells (Lee and Park, 1991). These results are in accordance with those of Saftner and Baldi (1990), in which the concentration of polyamines declined, while ethylene production increased during tomato fruit ripening. Polyamines also inhibited ethylene production in tobacco suspension culture cells and apple fruit discs by reducing the activities of the ethylene biosynthetic enzymes - ACC synthase and ACC oxidase (Apelbaum et al" 1981; Lee and Park, 1988). In tomato pericarp tissue, polyamines inhibited the accumulation of wound-inducible ACC synthase transcripts (Li et al., 1992). Furthermore, inhibitors of polyamine biosynthetic enzymes, such as MGBG ·or D-arginine, increased ethylene production and promoted senescence of cut carnation flowers, indicating that polyamines might inhibit ethylene production (Roberts et ai., 1984). However, the effects of polyamines on ethylene production in cut carnation flowers are controversial, since Downs and Lovell (1986) reported that exogenously applied putrescine and spermidine had no effects on ethylene production or senescence of cut carnation flowers. In this paper, we studied the effects of polyamines on ethylene biosynthesis using spermine, tetraamine, and MGBG.

Determination ofEndogenous Polyamine Content Polyamines were analyzed as described by Goren et aI. (1982). After dansylation, the dansylated polyamines were separated on TLC in chloroform: triethylamine (25: 2, VIV) and quantified using a spectrophotofluorimeter (F-2000, Hitachi, Japan), in which the emission at 500 nm was recorded afrer excitation at 350 nm.

RNA Extraction and Analysis Total RNA was extracted from carnation petals with guanidine HCI as described by Sambrook et aI. (1989). Twenty micrograms of the total RNA were separated e1ectrophoretically on a I % agarose gel containing 2.2 mollL formaldehyde, transferred to positively charged nylon membranes (Hybond-N+, Amersham, En~and), and alkaline-fixed. Membranes were hybridized with 5 x 10 cpm mL -1 denatured 32P-labeled cDNA probes of pSR120 and pCARACC403, which are an ACC oxidase cDNA clone (Wang and Woodson, 1991) and an ACC synthase cDNA clone (Park et aI., 1992), respectively. Equal amounts of loading were tested by ethidium bromide staining and by hybridizing the filters to a probe for SAM synthetase derived from carnation (Larsen and Woodson, 1991) (data not shown).

Results Materials and Methods

Plant Materials and Treatment ofSpermine and MGBG Flowers were harvested from greenhouse-grown carnations (Dianthus caryophyllus L. cv. Santa Pola) at anthesis and recut under water to overall lengths of IS cm. They were placed upright in flasks with stems in deionized water (DI) (contro!), or treatment solutions, and placed at 24 ± I·C under cool white fluorescent light (5,000 lux, 16 h light, 8 h dark). Treatments consisting of I mmollL spermine, solution per I mmollL MGBG, solution, were carried out for up to 6 days from anthesis.

Ethylene Measumnent

Ethylene Production and Polyamine Levels during Senescence In the petals of the control flowers, ethylene production was below detection until the 3rd day, but rapidly increased thereafter to the 6th day (Fig. 1) and then decreased on the

8

Petals were separated from the outer whorl of the flower and enclosed in a 100 mL gas-tight glass container and incubated at 24 ± I ·C under cool light for I h. A one mL sample of gas was withdrawn and analyzed with a gas chromatograph (GC-3BF; Shimadzu, Japan) equipped with an activated alumina column and a flame-ionization detector at 100·e. The data were mean values from 6 independent experiments. Error bars indicate SDs.

ACC Synthase Assay ACC synthase activity was analyzed as described by Woodson et aI. (1992). The enzyme sources were partially purified by a Sephadex G-50 column (0.7cmx7cm). ACC synthase activity was assayed by incubating 0.4 mL of enzyme source with 0.1 mL of 500 JlmollL SAM at 30·C for 15 min. The produced ACC was quantified by chemical conversion of ACC into ethylene.

In vivo ACC Oxidase Assay Assay of ACC oxidase activity was performed in vivo as described by Wang and Woodson (1989). ACC oxidase activity was determined by ethylene production from carnation petals infiltrated with I mmollL ACe.

Determination ofEndogenous ACC Content ACC contents were determined through chemical conversion of ACC to ethylene as described by Wang and Woodson (1989).

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Fig. 2: Changes in polyamine levels from petals of carnation flowers that were held with their stems in OI (0--0), 1 mmol/L spermine (e--e), or 1 mmollL MGBG (_--_) through 6 days after anthesis. (A): spermidine levels; (B): spermine levels. The data were mean values from 4 independent experiments. SO values (not shown) were estimated to be within 10% of the mean values.

7th day (data not shown). The levels of spermidine and spermine reached maximum at the 4th day in the control flowers (Fig. 2), which increased by about 160% and 140%, respectively, compared with the levels at the starting point of incubation. However, the levels of spermidine and spermine decreased concurrently with the increase of ethylene production from the 5th day (Figs. 1 and 2).

MGBG-treated flowers was 1.5 times higher than that of control at peak. One mmollL MGBG-treated flowers wilted from the 5th day and dried up at the 6th day, which was 1 day earlier than the control flowers (data not shown). From these results, we can conclude that MGBG advances senescence by 1 day through an increase in ethylene production. The fluctuation pattern of the changes in polyamine levels from the petals treated with spermine or MGBG was similar to that from the petals of control flowers (Fig. 2). However, the time points when the levels of polyamines reached their maximum were different from one another. The levels of spermidine and spermine peaked at the 5th day in sperminetreated petals, at the 3rd day in MGBG-treated petals, and at the 4th day in control petals. In the petals of MGBG-treated flowers, the increase in ethylene production correlated to the decrease in the levels of spermidine and spermine after the 3rd day (Figs. 1 and 2).

Effects o/Spermine and MGBG on Ethylene Biosynthetic Enzyme Activity

Endogenous free ACC content in the petals of the control flowers increased concomitantly with the increase of ethylene production (Fig. 4A). Free ACC content in the petals of control flowers was not detected in the early incubation period, but increased continuously after the 4th day until the 6th day of incubation (Fig. 4 A). In the petals of spermine-treated Effects o/Spermine and MGBG on Ethylene Biosynthesis flowers, free ACC was almost below detection during the enControl flowers wilted rapidly from the 6th day when the tire incubation period, corresponding to the low rate of ethylrate of ethylene production reached its maximum (Fig. 1). ene production (Fig. 1). Free ACC was not detected before They were dried up by the 8th day, whereas 1 mmol/L sper- the 6th day in the petals of MGBG-treated flowers, which mine-treated flowers did not show any significant wilting at produced much more ethylene than the controls (Fig. 4A). the 8th day (Fig. 3). The petals of spermine-treated flowers ACC synthase activity in the petals of control flowers bedid not produce any detectable ethylene before the 6th day came detectable after the 3rd day of incubation and increased (Fig. 1). Meanwhile, treatment with MGBG caused ethylene continuously to the 6th day (Fig. 4 B). The petals of MGBGproduction from the 3rd day when ethylene production in treated flowers showed an abrupt increase in ACC synthase the petals of control flowers was not detected at all. MGBG activities after the 3rd day. ACC synthase activity in MGBGincreased ethylene production, which reached its maximum treated petals was higher by almost two times that in the conlevel, 7.2 nL· g-I Fw· h -I, at the 5th day, and was earlier than trol petals at the 4th day. ACC synthase activity in sperminethe control by 1 day. Ethylene production in the petals of treated petals was not detected until the 6th day, and had

71

Effects of Spermine on Ethylene Biosynthesis

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Fig.4: Changes in ethylene biosynthetic enzyme activities and ACC contents in the petals of carnation flowers that were held with their stems in OI (0--0), 1mmollL spermine (e--e), 1 mmollL MGBG (0--0), or both 1 mmollL spermine and 1 mmollL MGBG (_--_) through 6 days after anthesis. (A): ACC contents; (B): ACC synthase activity; (C): ACC oxidase activity. The data were mean values from 4 independent experiments. Error bars indicate 50s.

only 20 % of the activity measured in the petals of control flowers. Petals treated with both 1 mmollL spermine and 1 mmollL MGBG had low ACC synthase activity, similar to that of petals treated with 1 mmol/L spermine only, which was much lower than control. ACC oxidase activity increased rapidly from the 4th day to the 6th day of incubation in the petals of control flowers (Fig. 4 C). In the petals of MGBG-treated flowers, ACC oxidase activity also increased rapidly from the 4th day. At the 5th day, it reached its maximum level with 2.8 times higher activity than that measured in control petals. The petals of spermine-treated flowers showed low ACC oxidase activity during the entire incubation period. In the petals treated with both 1 mmollL spermine and 1 mmollL MGBG, ACC oxidase activity could hardly be detected, as in the petals treated with 1 mmol/L spermine only.

Northern Blot Analysis To ascertain whether the changes in ethylene production through ACC synthase and ACC oxidase activities originated from the changes in the amounts of transcripts for ACC synthase and ACC oxidase, we analyzed the steady-state levels of their transcripts in carnation petals (Fig. 5). pCARACC403

and pSR120, which were ACC synthase and ACC oxidase cDNA dones, respectively (Wang and Woodson, 1991; Park et aI., 1992) were used as probes. It was shown in panel A of Fig. 5 that ACC synthase transcripts were detected starting at the 5th day in the petals of control and MGBG-treated flow,ers. ACC synthase transcripts were hardly detected in treatment with spermine only or, with both spermine and MGBG, through the entire incubation period. After 6 days, ACC synthase transcripts of control and of MGBG-treated petals were more abundant than on the 5th day, but those of spermine-treated and spermine plus MGBGtreated flowers could still be detected at very low levels. The steady-state levels of the transcripts of MGBG-treated petals were considerably higher than those of control. ACC oxidase transcripts in the petals of the control flowers accumulated from the 5th day and reached their maximum at the 6th day (Fig. 5 B). ACC oxidase transcripts began to accumulate from the 4th day and reached maximum at the 6th day in the petals of MGBG-treated flowers, which was much higher than those in control. In the petals of spermine-treated and spermine plus MGBG-treated flowers, ACC oxidase transcripts were detected only at the 6th day with a lower level compared with those in control or in MGBG-treated flowers.

72

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FIg. 5: Accumulation of ACC synthase and ACC oxidase transcripts in the petals of carnation flowers that were held with their stems in DI (C), 1 mmollL spermine (5), 1 mmollL MGBG (M), or both 1 mmollL spermine and 1mmollL MGBG (5M) through 6 days after anthesis. (A): ACC synthase transcripts; (B): ACC oxidase transcripts. Numbers indicate the incubation day after harvest. Twenty micrograms of total RNA were loaded per lane.

Discussion

We investigated the effectS of polyamines on ethylene biosynthesis in carnation flowers. We used carnation cv. Santa Pola, which was similar in the pattern of climacteric ethylene production and senescence to carnation cv. White Sim, but the rate of ethylene production from cv. Santa Pola showed one-tenth the: amount of cv. White Sim (Fig. 1; Woodson et al., 1992). This might be ascribed to the lower activities of ACC synthase and ACC oxidase in the petals of cv. Santa Pola than in the petals of cv. White Sim (Fig.4). Because: ethylene production dramatically increased according to the decrease of spermidine and spermine levels in the control flowers (Figs. 1 and 2), it can be suggested that there exists a correlation between the climacteric increase in ethylene production and the decrease in polyamine levels.

Treatment of 1 mmollL spermine markedly extended the vase life of carnation flowers (Fig. 3), and barely produced ethylene, even in petals of the 6th day, which was the climacteric stage of the control flowers (Fig. 1). The accordance of high levels of endogenous polyamine with a low level of ethylene production has been previously reported (Saftner and Baldi, 1990). The pericarp of tomato, Lycopersicon esculentum Mill. cv. Liberty, which ripens more slowly and has a more prolonged keeping quality than tomato cv. Rutgers and cv. Pink Red, showed increased free polyamine levels and decreased climacteric ethylene production in the ripening stage as compared with the pericarps of tomatoes cv. Rutgers and Pink Red. Therefore, it is possible that the low level of ethylene biosynthesis and the delay of senescence results from the high levels of endogenous polyamines. The treatment of high concentrations of chemicals, 10 mmollL spermine or 10 mmollL MGBG, dried the stem in the early incubation period (data not shown). This may be the result of cytotoxic effects of these chemicals. The treatment of 1 mmol/L MGBG shortened the vase life of carnation flowers through the acceleration of senescence (data not shown), and gave rise to an increase in ethylene production and a decrease in accumulation of endogenous polyamines in 4-day-old petals (Figs. 1 and 2) . It was also reported that the treatments of other inhibitors of polyamine biosynthesis such as D-arginine, difluoromethylarginine and dicyclohexylamine promoted the onset of senescence and increased ethylene production in several plant tissues (Roberts et al., 1984; Gallardo et al., 1995; Park and Lee, 1990). When the SAMDC gene was introduced into potato with antisense orientation (Kumar et al., 1996), it showed extremely low levels of polyamines and an increased rate of ethylene production by up to 40-fold. Also, this transgenic potato showed dwarfness, highly branched stems, short internodes, small leaves and early senescence. Therefore, the increase of ethylene production and the onset of senescence may be due to the decrease of endogenous polyamine levels. Because ethylene climacteric production followed the decrease of polyamine contents after one or two days in control, and all treated flowers, it can also be suggested that endogenous polyamine suppressed ethylene production in carnation petals. The increases in ethylene production by climacteric control and by spermine- or MGBG-treated petals were associated with increases in the activities and in the abundance of mRNAs for ACC synthase and ACC oxidase (Figs. 1,4 and 5) . Also, spermine effectively inhibited MGBG-induced increases in the activities and abundances of transcripts for ACC synthase and ACC oxidase. Therefore, we are able to draw the conclusion that ethylene production might be transcriptionally regulated. On the 4th day we could not detect any accumulation of ACC synthase transcripts in the controls or MGBG-treated petals, even though their enzyme activities were detected. It was thought that this inconsistency was caused by our having used the senescence-related ACC synthase and ACC oxidase genes (Woodson et al., 1992) as the probes for hybridization. These probes may not have been recognized as the genes for ethylene biosynthesis from the premature petals. All plant tissues including preclimacteric carnation petals appear to pro-

Effects of Spermine on Ethylene Biosynthesis

duce a low basal rate of ethylene (Woodson et al., 1992). Flower petals respond to this low level of ethylene with a climacterically increased ethylene production by the expression of senescence-related genes for ethylene biosynthesis after a critical stage of responsiveness is reached. In support of this, carnation petals have been found to become more sensitive to ethylene with age as judged by the induction of senescencerelated gene expression and ethylene biosynthesis (Woodson et al., 1992). As this climacteric proceeds, endogenous polyamines are thought to interrupt autocatalytic ethylene production by inhibiting the activities and/or senescence-related gene expression for the enzymes of ethylene biosynthesis. Ethylene production of MGBG-treated petals increased highly as compared with control, but the free ACC level was very low during the senescence of carnation flowers. This result might be ascribed to the fact that cellular ACC converts very rapidly into ethylene by significantly increasing the activity and the amounts of transcripts of ACC oxidase in MGBG-treated petals. In the climacteric stage of the 6th day, the activity of ACC oxidase decreased rapidly, although the transcripts for ACC synthase and ACC oxidase have been significantly accumulated by MGBG (Fig. 4). This inconsistency might be a result of the protein of ACC oxidase being regulated at the levels of translation or posttranslation, or at a process of development. Also, spermine content in climacteric petals of the controls increased, in spite of decreased spermidine content at the 6th day. One possibility is that the increase in the conversion of spermidine into spermine, which might be a constitutive process, has a significant role as a nitrogen reservoir for further developmental events such as seed formation after the death of the flower. Acknowledgements

This work was supported by a grant from the Korean Science and Engineering Foundation (931-0500-033-2) to K.Y.P. We thank Dr. William R. Woodson for providingpSR120andpCARACC403, and Dr. Choong II Lee for carnation cv. Santa Pola. We also thank Dr. Woo Taek Kim for his critical reading of this manuscript.

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