Plant Growth Regul DOI 10.

1007/s10725-011-9605-y

ORIGINAL PAPER

Effect of 3-cyclopropyl-1-enlyl-propanoic acid sodium salt, a novel water soluble antagonist of ethylene action, on plant responses to ethylene
Raphael Goren Moshe Huberman Joseph Riov Eliezer E. Goldschmidt Edward C. Sisler Akiva Apelbaum

Received: 5 January 2011 / Accepted: 22 June 2011 Springer Science+Business Media B.V. 2011

Abstract A novel water soluble inhibitor of ethylene action, 3-cyclopropyl-1-enyl-propanoic acid sodium salt [(CPAS) Patent Application number: PCT/IL2008/000995, US Application number 61/144758, International publication number: WO 2009/010981 AI] was synthesized in a highly puried form, and its effect to retard various exogenous or endogenous ethylene-mediated processes was tested. The inhibitor was applied by loading, dipping or spraying. CPAS retarded some ripening processes in avocado, banana, and peach fruit, delayed abscission of citrus leaf explants, inhibited leaf epinasty in tomato seedlings, and prolonged the vase-life of carnation and petunia owers. The fact that CPAS is a solid, water soluble, non-phytotoxic, and odorless inhibitor of ethylene action renders it a promising candidate for pre- and post-harvest application in a wide rang of open growing environments. Keywords Abscission Avocado Banana Carnation Peach Petunia Ripening Senescence Tomato Abbreviations CPAS 3-Cyclopropyl-1-enyl-propanoic acid sodium salt

1-MCP NBD

1-Methylecyclopropene 2,5-Norbornadiene

Introduction Ethylene is a natural plant growth regulator involved in various developmental processes, in particular fruit ripening, abscission, and senescence (Abeles et al. 1992). Ethylene has adverse pre-harvest effects, such as the acceleration of apple fruit drop and maturity, and particularly adverse post-harvest effects, namely acceleration of senescence and ripening of a wide range of agricultural crops. Increased ethylene production during senescence and ripening accounts for most of the post-harvest losses of agricultural commodities. Therefore, antagonists of ethylene action are potentially benecial for agriculture use, since they protect the tissues from both endogenous and exogenous ethylene (Sisler et al. 1985). Ethylene antagonists inhibit the action of ethylene at the molecular level by blocking its receptor site (Sisler and Wood 1988; Sisler et al. 2003). Thus, application of these inhibitors may allow extending the harvest season of crops, improve the keeping quality and prolong the storability and shelf-life of fruit, owers, herbs, and leafy vegetables. Several antagonists of ethylene action have been described in the past. The heavy metal silver is an effective means for controlling ethylene responses (Beyer 1979; Veen and Overbeek 1989), but its toxicity limits its use. The compound 2, 5-norbornadiene (NBD) is also an effective inhibitor of ethylene responses (Sisler et al. 1985; Goldschmidt et al. 1993; Sisler and Serek 1999), however, its strong odor and corrosive nature renders it not useful for

R. Goren (&) M. Huberman J. Riov E. E. Goldschmidt A. Apelbaum Robert H. Smith Faculty of Agriculture, Food and Environment, Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Kennedy-Leigh Centre for Horticultural Science, The Hebrew University of Jerusalem, 76100 Rehovot, Israel e-mail: rgoren@agri.huji.ac.il E. C. Sisler Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA

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agricultural purposes. Of the inhibitors of ethylene action developed so far, 1-methylcyclopropene (1-MCP) is considered to be the most promising antagonist for commercial use (Sisler et al. 1996; Sisler and Serek 1997; Feng et al. 2000; Watkins 2006), but its gaseous nature limits its use. It can be applied only in sealed systems, and cannot be used for loading of cut owers or applied as a spray in the eld. The limitations in the use of 1-MCP have led to the development of 1-MCP formulated as a wettable powder (AgroFresh, Rohm and Hass, Philadelphia, PA). Upon hydration of the wettable powder, 1-MCP is released as a gas. This, however, does not allow to dene the actual amount of 1-MCP that reaches the target tissues, and therefore its efcacy is unpredictable (Burns 2008; E. Sisler personal communication). Generally, an excessive amount of this formulation is used in eld applications to obtain the desirable effects. This in turn may pose a threat to other nearby plants or crops. Although the wettable powder of 1-MCP has shown some promise in apple and pear fruit (Elfving et al. 2007; Villalobos-Acuna et al. 2010), other attempts to use it in the eld were unsuccessful (Burns 2008). There are only two recent reports demonstrating the utilization of non-volatile N,N-alkyl-(1-cyclopropenylmethyl)amine and related compounds to counteract ethylene responses in banana fruit (Sisler et al. 2009) and improve the longevity of ornamentals crops (Seglie et al. 2010). In the present paper we report the anti-ethylene effects of another novel water soluble non-volatile cyclopropene derivative, 3-cyclopropyl-1-enyl-propanoic acid sodium salt (CPAS), in various plant systems.

were dipped in tap water. At the end of treatment, the fruit were immediately transferred to glass jars, sealed, and exposed to 250 ll l-1 ethylene for 24 h. The treated fruit were then ventilated and stored in controlled room for ripening assessment (peel color, fruit rmness, and peduncle abscission). Each treatment was carried out in eight replicates, one fruit per replicate. Experiments were repeated at least two times. Banana fruit Untreated, mature green banana fruit (Musa acuminate Colla cv. Grand Nanie) were obtained from a local supplier. Various concentrations of CPAS in aqueous solutions containing 0.025% Tween-20 surfactant were applied to the fruit by brushing the skin with a soft brush. Controls were brushed with aqueous solutions containing only 0.025% Tween-20. After 18 h, the fruit were treated with 300 ll l-1 ethylene for 24 h and stored as above for ripening assessment (peel color and fruit rmness). Each treatment was carried out in eight replicates, one fruit per replicate. Peach fruit Peach fruit (Prunus persica cv. White Lady) were harvested during the early commercial harvesting season. Fruit were sprayed on the day of picking with various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.1% Kinetic surfactant (Helena Chemical Company, Memphis, TN)). After 24 h, half of the fruit were sprayed again. Fruit were stored as described above for ripening assessment (fruit rmness). Each treatment was carried out in 6 replicates, one fruit per replicate. Tomato seedlings Tomato seedlings (Solanum lycopersicum L. cv. 1402) were obtained from a local nursery. The seedlings were transplanted into 600 ml containers containing peat-based medium and grown in a greenhouse. Three weeks after transplanting, seedlings with 67 leaves were sprayed with various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.1% Kinetic surfactant. Twenty four h after treatment, four seedlings per treatment, were treated with ethylene (3 ll l-1) in sealed jars for 24 h. Leaf epinasty was assessed by measuring the angle between the leaf petiole and the stem. Each replicate consisted of one seedling per replicate. Citrus leaf explants Citrus leaves (Citrus sinensis L. Osbeck cv. Shamouti) were obtained from mature trees growing in a local

Materials and methods Petunia and tomato seedlings were growing in a greenhouse until they reached the right developmental stage for the uptake and spraying experiments. At this stage the plants were transferred to control room (22C, 80% humidity and under standard uorescent light 16/8 day/ night). The other experiments, as described herein were treated and incubated at the same conditions. Avocado fruit Avocado fruit (Persea americana Mill cv. Hass) with long peduncles were harvested in a local plantation during the commercial harvesting season. For treatment with the inhibitor, the distal end of the intact peduncles were trimmed under tap water to a length of about 6 cm and dipped in aqueous solutions containing different concentrations of CPAS (patent No. WO 2009/010981 AI) for 30 h. Controls

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orchard. Leaves were picked in the morning, placed in plastic bags, and immediately treated upon arrival at the laboratory. Two-cm-long explants, each consisting of 1 cm petiole and 1 cm leaf blade tissues, were excised (Wurzburger and Goren 1978). The explants were treated with different concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.025% Tween-20 for 30 s. Twenty h after the inhibitor application, the explants were treated with 20 ll l-1 ethylene for 24 h. During and after the treatment, the explants were incubated in the controlled room. The rate of explant abscission was recorded daily. Each treatment was carried out in three replicates, 40 explants per replicate. Carnation owers and ower petals Carnation owers (Dianthus caryophyllus L. cv. White Sim) were harvested at the full bloom stage, when the outer petals deected at right angles to the stem. The owers were selected for uniformity in terms of ower diameter and placed in tap water. For experiments with cut owers, the owers were sprayed with various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.1% Kinetic surfactant. Twenty four hours after the inhibitor application, the owers were treated with 3 ll l-1 ethylene for 24 h. During treatment and vase-life, the owers were held in the controlled room. Each treatment was carried out in three replicates, four owers per replicate. For experiments with detached petals, uniform petals were excised from the outermost whorl of freshly harvested owers. The petals were immediately loaded with various concentrations of the inhibitor in 10 mM K-phosphate buffer, pH 7.6, in 2.5 ml vials for 18 h by immersing their cut bases in the treatment solutions. At the end of the inhibitor treatment, the petals were transferred to tap water and exposed to 5 ll l-1 ethylene for 24 h. The petals were then ventilated and incubated in tap water for daily observations of ower senescence, expressed as petal diameter. Each treatment was carried out in eight replicates, one petal per replicate. During and after treatment both the cut owers and the petals were incubated in the control room. Petunia owers Petunia owers (Petunia hybrida cv. Surnia White) were cut with a 57 cm pedicel at full bloom and placed in 5 ml tubes. The owers were treated with different concentrations of CPAS and then with ethylene as described for carnation petals. Flower diameter was used to assess ower senescence. Each treatment was carried out in six replicates, one ower per replicate.

Assessment of color change Skin color in avocado and banana fruit was assessed visually, using a scale from 0 (no color change) to 5 (complete change). Avocado: 0 = green, 5 = gray-black; banana: 0 = green, 5 = yellow. Measurement of rmness Fruit rmness was determined using a Chatillon digital force gauge (model DFG-50) tted with a 6 mm diameter conical probe. The rmness of each fruit was measured by inserting the conical probe through the fruit skin at four points around each fruit. Firmness data are expressed in either Newton (N) for avocado and banana or pounds (lb) for peach. Statistics Each experiment was repeated at least twice and one representative experiment is presented. Vertical bars are means plus or minus SE. Bars with different letters are signicantly different at P \ 0.05. The signicance level was analyzed using JMP program.

Results During the course of the present study we assessed the potency of CPAS to counteract the effects of exogenous and endogenous ethylene in various plant systems. In the experiments with exogenous ethylene, the inhibitor was applied before the ethylene treatment. It should be noted that in order to solely elucidate the potency of CPAS, no other treatments, such as fungicide application, were applied to any of the plant systems tested. Effect of CPAS on ethylene-induced ripening of avocado fruit Untreated avocado fruit ripened 34 days after the initiation of the experiment in terms of color (4.5 on the color index scale) and rmness (20 N) (Fig. 1a). CPAS signicantly protected the fruit against ethylene at concentrations of 20 mg l-1 and above. The retardation of color change by CPAS was concentration dependent, whereas softening retardation was similar in all the active concentrations of the inhibitor. Ripening of CPAS-treated fruit in terms of rmness occurred after about 8 days, while ripening in terms of color change occurred after 913 days, depending on the inhibitor concentration. Fig. 1b demonstrates that CPAS (100 mg l-1) delayed changes of fruit color and peduncle abscission at day 12 after treatment.

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16 14 12
Color Firmness

a
a a

Color index (Green =1 Black = 5)

A
5 4

B
A 12
10 8 6 4

One spray
Days after harvest 2 3

a
Two sprays

Days

10 8 6 4 2 0 0 0.8 4

a
3 2 1 0

a
B
8 6

Firmness (lb)

b b

Days after harvest 3 4

Firmness (lb)

20

100

500

100

b a b

a a a a

CPAS (mg l-1)

CPAS (mg l-1)

4 2 0

b b

Fig. 1 Effect of CPAS on ethylene-induced ripening and peduncle abscission of avocado fruit. Fruit, harvested with a long peduncle, were dipped in aqueous solutions containing various concentrations of CPAS for 30 h. The treated fruit were then exposed to 250 ll l-1 ethylene for 24 h at 22C. Control fruit were treated as described above without CAPS. a Time in days required to reach ripening (rmness20 N; peel color4.5 on a scale of 5). b Fruit peel color and peduncle abscission 10 days after treatment. Vertical bars indicate plus or minus SE of the means (n = 8). Bars with different letters are signicantly different at P \ 0.05

2 0

0 10 200 CPAS (mg l-1)

0 10 200 CPAS (mg l-1)

18 16 14 12 10 8 6 4 2 0

Color Firmness

5 4

b a ca
8 40 200 500

Color index (Green =1 Yellow = 5)

20

Fig. 3 Effect of CPAS on delaying ripening of peach fruit. Fruits immediately after harvest were sprayed with various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, plus 0.1% Kinetic. After 24 h, half of the treated fruit were sprayed again with the same CPAS solutions. Thereafter, the fruit were held in air at 22C for the required time. Fruit rmness after one (a) or two treatments (b). Vertical bars indicate plus or minus SE of the means (n = 8). Bars with different letters are signicantly different at P \ 0.05

Days

2 1 0

Effect of CPAS on delaying ripening of peach fruit Fruit rmness at harvest was 812 lb. Peach fruit are unmarketable when their rmness is below 4 lb. Control fruits softened quickly and were unmarketable 2 days after harvest (Fig. 3). The effect of the inhibitor on fruit softening was examined by spraying it once, immediately after harvest, or twice, an additional spray 1 day after harvest. One spray of CPAS at concentrations of 10 and 200 mg l-1 signicantly retarded softening 2 days after harvest (Fig. 3a). Although the softening of the treated fruit increased signicantly 3 days after harvest, they were still marketable at this date. The two inhibitor concentrations did not differ much in their effect on fruit softening during the above period. Two sprays of CPAS kept fruit marketable 4 days after harvest, again with little difference between the two concentrations applied (Fig. 3b). Effect of CPAS on ethylene-induced leaf epinasty in tomato seedlings Ethylene treatment induced a signicant epinasty of tomato leaves measured 24 h after treatment (Fig. 4a, b). CPAS somewhat counteracted the ethylene effect at 40 mg l-1 concentration, but its inhibitory effect was more signicant at 80 and particularly at 160 mg l-1, which completely counteracted the ethylene effect (Fig. 4a, b).

ca
0

ca
1.8

CPAS (mg l-1)

0 200 CPAS (mg l-1)

Fig. 2 Effect of CPAS on ethylene-induced ripening of banana fruit. Fruit were brushed with various concentrations of CPAS in tap water plus 0.025% Tween-20. Control fruit were brushed with water plus Tween-20 without CPAS. After 18 h, the fruit were treated with 300 ll l-1 ethylene for 24 h at 22C, ventilated and held in air for the required time. Controls were treated with the same treatments solution without CAPS. a Time in days required to reach ripening (rmness 15 N; peel color4.5 on a scale of 5). b Fruit peel color 18 days after treatment. Vertical bars indicate plus or minus SE of the means (n = 8). Bars with different letters are signicantly different at P \ 0.05

Effect of CPAS on ethylene-induced ripening of banana fruit Untreated banana fruit ripened 3 days after exposure to ethylene in terms of color change (4.5 on the color index scale) and rmness (15 N) (Fig. 2a). CPAS signicantly retarded the effect of ethylene on the color change already 8 days after treatment at 40 mg l-1 concentration. Fig. 2b demonstrates CPAS treated fruits (200 mg l-1) 18 days after treatment. The inhibitor had almost no effect on the ethylene-induced softening at all the concentration range examined.

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Air

A
Leaf petiole degree from stem

100 80 Abscission (%) 60 40 20

100 80 60 40 20 0

Ethylene

a a a

CPAS + Ethylene

ab b b

10

20

40

80

160

CPAS (mg

l-1)

16

40 68 Incubation time (hours)

92

Angle

Fig. 5 Effect of CPAS on ethylene-induced abscission of citrus leaf explants. Leaf explants were immersed in various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.025% Tween-20, for 30 s, incubated in air for 24 h and then exposed to 20 ll l-1ethylene for 24 h at 22C. Control explants were treated as described above without CAPS. Numbers in parenthesis depicts CPAS concentration. Vertical bars indicate plus or minus SE of the means (n = 10)
0 0 40 80 160

Air

CPAS (mg

l-1)

Fig. 4 Effect of the CPAS on ethylene-induced leaf epinasty of tomato seedlings. Three-week-old tomato seedlings (with 67 leaves) were sprayed with various concentrations of CPAS in 20 mM Kphosphate buffer, pH 7.6 and 0.1% Kinetic, incubated for 24 h at 22C in air and then exposed to 3 ll l-1 ethylene for 24 h at 22C. Control seedlings were treated as described above without CAPS. a Change in leaf angle, immediately after ethylene treatment (degrees, see arrow in b). b Tomato seedlings after the ethylene treatment. Vertical bars indicate plus or minus SE of the means (n = 16). Bars with different letters are signicantly different at P \ 0.05

of the inhibitor, 3 and 9 mg l-1, did not have any effect on ethylene-induced ower senescence. All the higher inhibitor concentrations examined signicantly retarded ower senescence (Fig 6a, b), with some advantage of the highest concentration. Flower treated with these concentrations were still marketable 6 days after the initiation of the experiment (Fig. 6b). Effect of CPAS on ethylene-induced senescence of excised carnation petals Senescence of excised carnation petals is manifested by inrolling of the petals, which can be quantied by measuring the diameter of the petals. Ethylene treatment induced rapid senescence of inhibitor-untreated petals, which was already evident 2 days after the initiation of the experiment (24 h air ? 24 h ethylene) (Fig. 7a). The lowest concentration of the inhibitor, 3 mg l-1, did not differ from the control, but higher concentrations were effective in counteracting the effect of ethylene in a concentration dependent manner. Petals treated with the two highest concentrations of CPAS, 27 and 81 mg l-1, showed only a partial in-rolling even after 14 days. Figure 4b demonstrates the retarding effect of CPAS (81 mg l-1) at day 10 after treatment. Effect of CPAS on ethylene-induced senescence of excised petunia owers Ethylene treatment accelerated senescence of inhibitoruntreated owers, resulting in complete senescence 5 days after treatment. CPAS counteracted the ethylene effect in a concentration dependent manner. The lowest concentrations,

Effect of the CPAS on ethylene-induced abscission of citrus leaf explants Inhibitor-untreated citrus leaf explants exhibited about 70% abscission 40 h (16 h air ? 24 h ethylene) after the initiation of the experiment, and reached 100% abscission after 68 h (Fig. 5). The inhibitor retarded abscission in a concentration dependent manner. Some retardation was already evident with the lowest concentration (7 mg l-1) examined, and explants treated with the highest concentration, 189 mg l-1, reached only about 55% abscission after 92 h (Fig. 5). Effect of CPAS on ethylene-induced senescence of carnation cut owers Inhibitor-untreated cut carnation owers senesced completely 2 days after the initiation of the experiment (24 h air ? 24 h ethylene) (Fig. 6a). The two lowest concentrations

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100 90

Diameter (mm)

80 70 60 50 40 0 0 1 2 3 4 5 6

Incubation time (days)

Flower diameter

27

90

270

CPAS (mg l-1)

Fig. 6 Effect of CPAS on ethylene-induced senescence of carnation cut owers. Flowers at full bloom were sprayed with various concentrations of CPAS in 20 mM K-phosphate buffer, pH 7.6, containing 0.1% Kinetic, incubated in air for 24 h at 22C and then exposed to 3 ll l-1 ethylene for 24 h at 22C. Control owers were treated as described above without CAPS. After treatment the cut owers were held in water for observations. a Flower diameter 5 days after treatment (numbers in parenthesis depicts CPAS concentration). b Flowers at the 4th day after treatment. Vertical bars indicate plus or minus SE of the means (n = 6)

0.1 mg l-1, signicantly delayed senescence up to 9 days after initiation of the experiment (24 h air ? 24 h ethylene) (Fig. 8A). Flowers treated with higher concentrations (6 and 24 mg l-1) underwent minor senescence after 11 days, and owers were treated with the highest concentration (72 mg l-1), retained their decorative value even after 18 days. Figure 8b demonstrates the retarding effect of CPAS (72 mg l-1) at day 12 after treatment.

Discussion Although the need for a water soluble inhibitor of ethylene action for agricultural use is quite clear (Burns 2008),

attempts made in this direction have yielded only very few promising compounds. Besides 1-MCP formulated as a wettable powder, only some water soluble dialkylamine derivates of 1-MCP have been demonstrated to have a potential for agricultural use (Sisler et al. 2009, Seglie et al. 2010). The present report demonstrates the activity of a newly synthesized ethylene action inhibitor, CPAS. This is the rst time that a water soluble cyclopropene derivative was synthesized in a solid, highly puried ([90%) form. This allowed us to determine the exact concentrations applied in the various experiments. CPAS counteracted the effect of exogenous and endogenous ethylene in various ethylene-mediated processes: color change of avocado and banana fruit during ripening, softening of peach fruit, epinasty of tomato leaves, abscission of citrus leaf explants; and senescence of carnation and petunia cut owers. It also retarded epinasty and abscission of cotton leaves, and ethylene-induced chlorophyll degradation of wheat cotton and tobacco leaves, as well as to increasing grain yield of wheat (Goren et al. in preparation). These data demonstrate that the inhibitor can be utilized for agricultural use in open spaces, such as elds, plantations, greenhouses and other unsealed growing and storage facilities, where 1-MCP cannot be used efciently. Although we did not compare the activity of CPAS to its volatile form (the free acid) or to 1-MCP in the present research, data of a previous study showed that the more hydrophobic the inhibitors are, the more active they might be (Sisler et al. 2003). Hence, water solubility seems to make the inhibitor less active. Indeed, our data show that the effect of CPAS was usually obtained in concentrations of dozens or hundreds ppm, much higher than those of 1-MCP. Nevertheless, Sisler et al. (2009) suggested that a hydrophobic attachment between water soluble inhibitors and the ethylene receptor forces the inhibitor molecule out of the water into the cell membrane, where the receptor is located, enabling the inhibitor to bind to the receptor. Another reason for the reduced activity of salts compared to their corresponding volatile forms might be lower penetration and diffusion rates within the tissues (Seglie et al. 2010). This also has a practical drawback, particularly with fruit. While reduced penetration can be effectively overcome by the use of surfactants, as was done in the present work, diffusion of salts into bulky tissues might be limited. This is demonstrated by the present (Figs. 1 and 2) and previous (Sisler et al. 2009) data, that while fruit color change during ripening was signicantly delayed by the water soluble inhibitors, fruit softening was hardly affected. Peach was somewhat exceptional to this phenomenon due to its thinner skin. Loading of carnation cut owers with CPAS solutions was ineffective (data not shown), whereas application of the

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36 30

A
Petal width

B
Petal width (mm)
24 18 12 6 0 0 2 4 6 8 10 12 14

81

Incubation time (days)

Fig. 7 Effect of CPAS on ethylene-induced senescence of excised carnation petals. Petals from the outermost whorl of the ower were excised and loaded with different concentrations of CPAS in 10 mM K-phosphate buffer, pH 7.6, for 18 h at 22C and then exposed to 5 ll l-1 ethylene for 24 h at 22C. Control petals were

CPAS (mg

l-1)

treated as described above without CAPS. After treatment the petals were held in tap water for observations. a Flower petal diameter during 14 days after treatment (numbers in parenthesis depicts CPAS concentration). b Flower petals 10 days after treatments. Vertical bars indicate plus or minus SE of the means (n = 10)

B
80

64 48 32 16 0 0 3 6 9 12 15 18
0 72

Incubation time (days)

CPAS (mg l-1)

Fig. 8 Effect of CPAS on ethylene-induced senescence of petunia cut owers. Flowers at full bloom with a 57 cm-long pedicel were loaded with different concentrations of CPAS in 10 mM K-phosphate buffer, pH 7.6, for 18 h at 22C and then exposed to 5 ll l-1 ethylene for 24 h at 22C. Controls were treated as described above without CAPS. After treatment, the cut owers were held in water for observations. a Flower diameter 18 days after treatment (numbers in parenthesis depicts CPAS concentration). b Flowers 12 days after treatments. Vertical bars indicate plus or minus SE of the means (n = 6)

inhibitor by spraying the owers was highly effective in delaying senescence (Fig. 6). These data indicate that CPAS can not move freely in the xylem. Similar observations, which have been reported for various organic compounds, including plant hormones and herbicides, are related to uptake by adjacent cells or more likely to adsorption onto cell wall components, particularly lignin (Abebie 2008 and literature cited therein). It should, however, be mentioned that the inhibitor was successfully applied by loading in

most of the other plant systems used in the present study, including petunia owers, as well as in excised petals of carnation open owers, and citrus leaf explants. Although CPAS inhibited various ethylene-induced processes, the concentration range necessary to exert signicant effects varied greatly between species. For example peach ripening was already delayed at a much lower concentration (Fig. 3) compared to that needed to inhibit peel color change of avocado and banana fruit (Figs. 1, 2). Similar results were reported by Feng et al. (2000, 2004) and by Seglie et al. (2010). As mentioned above, the reason for that might be a different penetration and particularly diffusion rate. The different response of various species might also result from differences in the catabolism of the inhibitor or the binding sites characteristics. The latter include the abundance of the binding sites, the rate of synthesis of new binding sites, and the rate of dissociation of the inhibitor from the binding sites. In conclusion, although CPAS, in its water soluble form has some drawbacks compared to volatile ethylene action inhibitors, the possibility to apply it in a wide range of open growing environments makes it a promising potential ethylene inhibitor for agricultural use. The present data clearly demonstrate that whenever CPAS reached the target site it proved to be an effective ethylene action antagonist. Moreover, within the concentration range examined in the various plant systems used, no phytotoxic symptoms were observed, and at the end of the antagonist delaying effect by CPAS the treated fruit ripened normally.
Acknowledgments This research was supported by Research Grant No. IS-3493-03CR from BARD, The United States-Israel Bi-national US-Israel Agricultural Research and Development Fund.

Flower diameter (mm)

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