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Abstract
We investigated the effect of a preservative solution containing boric acid on the senescence of cut carnation flowers
(Dianthus caryophyllus L. cv. Master). A 24-h pulse treatment with the preservative solution containing 50, 75 or 100
mM boric acid or continuous treatment with 1 mM boric acid resulted in strong inhibition of the climacteric ethylene
production. Both the pulse and continuous treatments significantly increased flower longevity. Free and conjugated
1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase activity increased in carnation petals during
senescence, although significantly less in boric acid-treated carnations than in control flowers. The levels of putrescine
increased as senescence progressed in both control and boric acid-treated carnations and an increase in spermidine
levels was higher in treated carnations. Abscisic acid levels in petals also increased during senescence, but much less
in boric acid-treated carnations. It is concluded that boric acid prevents the early rise in ethylene production and
considerably improves carnation vase life. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Abscisic acid; Boric acid; Dianthus caryophyllus; Ethylene; Polyamines; Senescence
0925-5214/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 5 - 5 2 1 4 ( 0 1 ) 0 0 1 0 8 - 9
134 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142
2.2. Ethylene production and respiration rate were cut into small pieces and enclosed in vials
with 25 mM Tris– Hepes buffer, pH 7.5, contain-
To measure ethylene production and respira- ing 1 mM ACC. After 2 h at 30°C and continuous
tion rate, cut carnations were individually en- shaking, a 1-ml gas sample of the vial atmosphere
closed in 500 ml glass jars fitted with a silicon was withdrawn and monitored for its ethylene
septum for 1 h. After this time, a 1 ml sample of content. ACC oxidase activity was expressed as
the jar atmosphere was withdrawn and the ethyl- nanolitres of ethylene released per gram fresh
ene concentration determined using a Hewlett– weight per hour (nl g − 1 h − 1).
Packard model 5890 gas chromatograph
(Wilmington, DE) equipped with a flame ioniza- 2.5. Polyamine extraction and quantification
tion detector (FID) and a 3 m length stainless
steel column with 3.17 mm inner diameter, Polyamines were extracted with HClO4 and an-
filled with 80/100 activated alumina. Results alyzed by the benzoylation method, as previously
were expressed as nanoliters of ethylene produced reported (Serrano et al., 1991). Extracts for
per gram of fresh weight per hour (nl g − 1 h − 1) polyamine analysis were prepared by homogeniz-
and are the mean9S.E. of ten flowers. Another 1 ing six carnation petals in 10 ml of 5% HClO4
ml gas sample of the same jar was used to using a mortar and pestle. The homogenate was
determine CO2 concentration in a Shimadzu 14-B then centrifuged for 30 min at 20 000× g and the
gas chromatograph (Kyoto, Japan). Respiration supernatant was used to quantify the polyamine
rate was expressed as milligram of CO2 re- content in duplicate. A total of 2 ml of the
leased by a kilogram of fresh weight per hour (mg supernatant were mixed with 2 ml of 4 N NaOH
kg − 1 h − 1). Results are the mean9 S.E. of ten and 20 ml of benzoyl chloride in a glass tube.
flowers. After vortexing for 15 s, the mixture was incu-
bated for 20 min at room temperature. Saturated
2.3. ACC extraction and assay NaCl (4 ml) and 4 ml of cold diethyl ether
were then added. The tube content was vortexed
Total (free and conjugated ACC) was extracted for 15 s and incubated for 30 min at − 18°C.
as previously described (Serrano et al., 1991). Finally, 2 ml of the ether phase (containing
Fifteen petals were macerated in a mortar with a benzoyl-polyamines) were evaporated under nitro-
pestle in 10 ml of 0.2 M trichloroacetic acid. The gen and redissolved in 1 ml of methanol (HPLC
macerate was centrifuged at 7000×g for 10 min grade). Benzoyl-polyamines were analyzed by
and the supernatant was used to determine its free HPLC using a Hewlett–Packard system, series
ACC content by chemical conversion of ACC to 1100 (Waldbrom, Germany). The elution
ethylene which was then quantified as described system consisted of methanol:water (64:36, v/v as
above. Conjugated ACC was hydrolyzed to free solvent), run isocratically with a flow rate of 0.8
ACC with 2 N HCl. In both cases, measurements ml min − 1. The benzoyl-polyamines were
were made in triplicate. A relative calibration eluted through a reverse-phase column
procedure was used to determine the amount of (LiChroCart 250-4, 5 mm, Merck, Darmstadt,
ACC in samples using a standard curve of ACC Germany) and detected by absorbance at 254 nm.
from Sigma (Poole, Dorset, UK). Results were A relative calibration procedure was used to de-
expressed as nmol per gram fresh weight (nmol termine the amounts of polyamines in samples
g − 1 f.w.) and are the mean9 S.E. of triplicate using standard curves of putrescine, spermidine
measurements on each of three carnations. and spermine from Sigma and adding hexa-
nediamine as the internal standard. Results were
2.4. ACC oxidase acti6ity expressed as nmol per gram fresh weight (nmol
g − 1 f.w.) and are the mean9S.E. of two mea-
ACC oxidase activity was measured in triplicate surements made independently on three carna-
in each carnation flower. Three carnation petals tions.
136 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142
2.7. Statistics
ethylene. These findings are consistent with the on day 12 (Fig. 3B). However, in carnation flow-
proposal that petal in-rolling and withering are ers kept continuously in PS with 1 mM boric acid,
ethylene independent. They agree with the results ACC oxidase remained very low, only a small
of Satoh et al. (2000) and Onoue et al. (2000), increase being observed on day 16 (Fig. 3B).
who reported that the expression of ACC syn- These results show that the lack of ethylene pro-
thase and ACC oxidase genes and that of cysteine duction in boric acid-treated carnations could be
proteinase (related to withering of petals) were due to a combination of two causes: a low
regulated differently in carnation petals. availability of free ACC, probably resulting from
The continuous treatment with the PS contain- low ACC synthase activity, and the failure to
ing 1 mM boric acid was selected to study the convert ACC into ethylene because of decreased
effect of boric acid on ethylene, polyamines and ACC oxidase activity.
ABA biosynthesis. In untreated carnations, the ability of petal
tissue to convert exogenous ACC into ethylene in
3.3. ACC le6els and ACC oxidase acti6ity the presence of boric acid was assayed at two
developmental stages. Ethylene production in-
Free and total ACC levels were very low in creased with increasing ACC concentrations in
recently harvested flowers and rose slowly in con- both preclimacteric and climacteric carnations
trol flowers during the first 8 days of senescence at (Table 2). The higher ethylene production rate in
20°C (Fig. 3A). After day 8, free and total ACC climacteric petals shows that ACC oxidase activ-
levels rose sharply (Fig. 3A). In carnations main- ity increased during senescence (as can also be
tained in the PS with 1 mM boric acid, free and inferred from Fig. 3B). However, no significant
total ACC levels in the petals were significantly differences in ethylene production were found
lower than in control flowers (Fig. 3A). Although when 10 and 20 mM boric acid were added to the
ACC synthase was not directly measured, results incubation medium. This finding shows that boric
show that boric acid treatment probably inhibited acid does not directly inhibit ACC oxidase activ-
this enzyme by \50%, but did not inhibit ACC ity, as do cobalt ions and a-aminoisobutyric acid
conjugation, since only a small fraction of the (Serrano et al., 1990). Thus, the low ACC oxidase
ACC synthesized remained in a free state (Fig. activity and ACC levels found in boric acid-
3A). treated carnation petals during senescence may be
ACC oxidase activity in control carnations was due to the effect of boric acid on the synthesis of
very low during preclimacteric stages but in- both ACC synthase and ACC oxidase, which
creased along with ethylene production, peaking normally occurs during carnation senescence
Table 1
Longevity (days) of Master carnation flowers after pulse treatment with a pretreatment solution containing various concentrations
of boric acid or continuously kept in the solution containing 1 mM boric acida
0 25 50 75 100
Longevity 12.2 90.4a 16.79 1.0b 20.8 9 0.7c 21.7 9 0.7c 22.29 0.5c
Continuous treatment (boric acid (mM) concentration in the PS)
0 1
a
Each experiment consisted of ten flowers. Means within each treatment group followed by different letters are significantly
different (PB0.05).
M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142 139
Fig. 3. Free and total ACC content (A) and ACC oxidase
activity (B) evolution in petals from continuously-treated car- Table 2
nations with PS (control) and PS containing 1 mM boric acid, Ethylene production (nl g−1 h−1) of carnations petals at two
during senescence at 20°C. Data are the mean 9 S.E. of mea- stages of development after incubation for 2 h with various
surements made in triplicate in each of three flowers. ACC concentrations plus 10 or 20 mM boric acida
(Jones and Woodson 1999; Satoh et al., 2000). ACC Boric acid concentration (mM)
Another possibility is that the low ACC oxidase concentration
(mM) 0 10 20
activity could be due to substrate limitation (Fig.
3). Since ACC synthase and ACC oxidase activity Preclimacteric stage
are in an autocatalytic loop, the present data do 0.1 22.3 9 3.7a 19.1 93.1a 25.4 9 3.2a
not suggest which part of the chain is broken. 0.5 157.4 911.6b 132.6 912.9b 149.6 914.8b
1 258.1 912.9c 282.7 925.0c 294.0 921.5c
Climacteric stage
3.4. Polyamine le6els 0.1 229.9 919.2a 226.5 926.8a 204.0 919.0a
0.5 267.2 98.7b 285.4 922.2b 269.1 921.0b
The most abundant polyamines in the carna- 1 367.8 924.3 386.5 925.7c
c
361.1 917.0c
tion petals of the cultivar Master were putrescine a
Data are the mean 9S.E. of two replicates of three petal
and spermidine. Spermine levels were very low, samples per treatment. Means within each row and column
just at or below the detection limit. In control (for each development stage) followed by different letters are
flowers, putrescine levels increased slowly during significantly different (PB0.05).
140 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142
4. Conclusions
longevity and could be useful in the cut flower production in tomato fruit. Phytochemistry 43, 323 –
industry. Boric acid treatments apparently inhib- 326.
Odom, R.E., 1954. Research on the keeping of out flowers.
ited ethylene production in carnation flowers by Mededelingen Directeur van de Tuinbouw 17, 830 – 836 In
lowering ACC synthase activity and consequently Dutch with a summary in English.
the availability of ACC and probably also by Onoue, T., Mikami, M., Yoshioka, T., Hashiba, T., Satoh, S.,
inhibiting the synthesis of ACC oxidase. In addi- 2000. Characteristics of the inhibitory action of 1,1-
tion, spermidine levels were higher in the boric dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on ethyl-
ene production in carnation (Dianthus caryophyllus L.)
acid treated carnations, whereas the increase of flowers. Plant Growth Reg. 30, 201 – 207.
ABA levels was lower. Thus, increased spermidine Peiser, G., 1986. Levels of 1-aminocyclopropane-1-carboxylic
levels and lower ABA levels may also be responsi- acid (ACC) synthase activity, ACC, and ACC-conjugate in
ble for the inhibition of ethylene production of cut carnation flowers during senescence. Acta Hortic. 181,
boric acid treated carnations. 99 – 104.
Podd, L.A., Van Staden, J., 1999. Is acetaldehyde the causal
agent in the retardation of carnation flower senescence by
ethanol? J. Plant Physiol. 154, 351 – 354.
Acknowledgements Reid, M.S., Wu, M., 1992. Ethylene and flower senescence.
Plant Growth Reg. 11, 37 – 43.
This study was funded by the Comisión Inter- Roberts, D.R., Walker, M.A., Thompson, J.E., Dumbroff,
E.B., 1984. The effect of inhibitors of polyamine and
ministerial de Ciencia y Tecnologı́a (CICYT),
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project PETRI-95-0271-OP. and polyamine levels in cut carnation flowers. Plant Cell
Physiol. 25, 315 – 322.
Satoh, S., Kosugi, Y., Iwazaki, Y., Shibuya, K., Waki,
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