Physiological Characterization of Flower Senescence in Long Life and

Ephemeral Hibiscus (Hibiscus rosa-sinensis L.)

A. Trivellini and P. Vernieri A. Ferrante G. Serra
Dip. Biologia delle Piante Agrarie Dip. Produzione Vegetale Scuola Superiore S. Anna
University of Pisa University of Milan Pisa
Italy Italy Italy

Keywords: ABA, anthocyanins, carotenoids, ethylene, phenols, post-production

Abstract
The most part of hibiscus plants produces short life flowers that last one day.
Therefore they are called ephemeral and flower senescence is usually associated with
petal in-rolling and abscission. Flowers are well shaped and different organs can be
easily separated, these characteristics make the hibiscus a good model system for
flower senescence studies.
The aim of this work was to identify metabolic differences and hormonal
profiles in petals of different flowers of hibiscus plants that show different flower
longevity. Different hibiscus (Hibiscus rosa-sinensis L.) plants were screened under
controlled environment for identifying different clusters. Among the plants screened
the physiological studies were performed on the following cultivars: Caribbean
Tricolour, Caribbean Dark Pink, Caribbean Pink, Caribbean White,
Caribbean White eye, Porto and La France. Flowers were detached from
different clusters and placed in postharvest room for vase life determination. Flower
harvesting was performed in the morning usually between 9:00 and 12:00 a.m.
Detached flowers were placed in deionised water. Ethylene production, abscisic acid
content, anthocyanins, total phenols and carotenoids were measured. All data were
analysed and correlated with flower longevity. The ephemeral flowers lasted 12-18 h,
while the longest flower life was 3-4 days in Porto. The ethylene production and
ABA content were inversely proportional to flower longevity. In fact the longest
flower life was observed in hibiscus plants that had the lowest ethylene production.

INTRODUCTION
Flowering potted plants are ornamental items that are characterised by a
cultivation period in optimal growing conditions. Subsequently, when plants reach the
desired commercial size, they can be transferred in hardening greenhouses or directly sent
to the distribution chain (Nowak and Rudnicki, 1990). Usually flowering potted plants are
packed with transparent films and stored or shipped to different selling destinations. The
ornamental quality of flowering potted plants depends on the number and quality of
flowers, mainly open flowers. Moreover, the nutrient management during cultivation is
also very important. It has been demonstrated that water stress combined with phosphorus
and nitrogen deficiency during cultivation improved the post-production quality (Hansen
et al., 2005). The presence of flowers on plants depends on the flower longevity and
turnover. Hibiscus plants usually produce flowers that last one day and they are classified
as ephemeral. Flowering hibiscus potted plants are very sensitive to exogenous ethylene
and its effect is visible at concentrations as low as 0.1 l L-1. Ethylene induces flower
senescence and in hibiscus plants causes buds abscission, compromising the ornamental
quality (Hyer, 1996; Hansen et al., 2005). Physiological studies during hibiscus flower
senescence showed that ethylene production and 1-aminocyclopropane-1-carboxylic acid
(ACC) content increased in petals during flower development and senescence (Woodson
et al., 1985). Exogenous applications of ACC or amino-oxyacetic acid (AOA) increased
or reduced ethylene production respectively, with positive or negative effect on flower
life. The ethylene production and accumulation during transportation or storage can have
dramatic effect on bud abscission reducing the commercialization quality (Al-Saqri et al.,
2003). Treatments with ethylene action ethylene inhibitors, such as 1-MCP or STS,

Proc. IC on Qual. Manag. Supply Chains of Ornamentals 457

Eds.: S. Kanlayanarat et al.
Acta Hort. 755, ISHS 2007
extended flower life of one day compared to the controls. The 1-MCP treatment was
effective only if applied continuously (Reid et al., 2002). Beside ethylene also abscisic
acid (ABA), another plant hormone, increases during hibiscus flower senescence
(Swanson et al., 1975). The endogenous levels of these two hormones may be linked with
genetic background of cultivars and might be used as markers for classic breeding
programs which have as target to extend flower life.
The aim of this work was to study the endogenous variations of ethylene
production, ABA content and flower pigments during flower senescence of different
hibiscus cultivars characterised by different flower longevity.

MATERIALS AND METHODS

Plant Material
Hibiscus (Hibiscus rosa-sinensis L.) plants were grown in greenhouse under
natural environmental conditions at the Dip. Biologia delle Piante Agrarie, University of
Pisa. The experiments were carried out on ephemeral (La France) and long life (Porto,
Caribbean White, Caribbean White eyes, Caribbean tricolour) hibiscus plants.
Flowers were harvested from May to July, period of the highest flower production,
between 9:00 and 12:00 a.m. Detached flowers were immediately transported to the
laboratory and placed in distilled water. All experiments were performed under controlled
environment conditions (20C T, 70% RH, 10 mol m-2 s-1 PPFD, 12 h photoperiod).
Flower life was determined in these conditions by daily observations. All measurements
were carried out on one day fully open flowers.

Ethylene Measurements
Ethylene production was measured by enclosing flower organs in air-tight
containers (250 ml). Two ml gas samples were taken from the headspace of the containers
after 1 h incubation at room temperature. The ethylene concentration in the sample was
measured by a gas chromatograph (HP5890, Hewlett-Packard, Menlo Park, CA) using a
flame ionization detector (FID), a stainless steel column (150 x 0.4 cm packed with
Hysep T), column and detector temperatures of 70 and 350C, respectively, and nitrogen
carrier gas at a flow rate of 30 ml min-1.
Abscisic Acid Determination
Petals (80-100 mg FW) were extracted with distilled water (water:tissue ratio 10:1
v/w) for 16 h at 4C in the dark. Quantitative analysis was performed on crude aqueous
extracts using a solid-phase radioimmunoassay based on a monoclonal antibody (DBPA1)
raised against free (S)-ABA, as described previously (Vernieri et al., 1991).

Determination of Carotenoids, Anthocyanins and Total Phenol Absorbance

Total carotenoids for hibiscus petals were determined by extraction using
methanol 99.9% as solvent. Petal samples were kept in a dark, cold room at 4C for 24 h.
Quantitative determination of carotenoids was carried out immediately after extraction.
Absorbance readings were performed at 470 nm. Total carotenoids were calculated using
Lichtenthalers formula (1987).
Petals of the frozen tissue (50 mg) were extracted into methanolic HCl (1%).
Samples were incubated overnight at 4C in darkness. The concentration of anthocyanins
was expressed in cyanidin-3-glucoside equivalents determined spectrophotometrically at
535 nm using an extinction coefficient of 29,600 ().
Phenolic compounds were extracted from 30-50 mg of petals using 5 ml 1.2 M
HCl in 99% methanol. Absorbance measurements were taken after overnight incubation
at 4C. Total phenolics were estimated by measuring absorbance at 320 nm using an UV-
Vis spectrophotometer (Ke and Saltveit, 1989). The amount of total phenols was
expressed as gallic acid equivalents.

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Statistical Analysis
The data are reported as mean values with a standard error (SE, n=5). The data
were subjected to one-way analysis of variance and the differences among treatments
were determined by Tukeys test (P<0.05).

RESULTS
The physiological investigations were performed in flowering potted hibiscus
grown in the same environment. The plant hormones and leaf pigments were determined
on detached flowers placed in controlled chamber. No differences in flower life were
observed between attached and detached ones. After 2 hours adaptation in the controlled
chamber all measurements were carried out. Ethylene production and ABA content
showed the same trend among cultivars (Figs. 1A, B). The highest values were found in
ephemeral flowers (2722 nl g-1 h-1) harvested from La France. On the contrary the
lowest ethylene values (543 nl g-1 h-1) were obtained from petals of Porto, which had
flowers with the longest life, 3 days on average. The ethylene biosynthesis of the
ephemeral hibiscus flowers (1 day) was 5-fold higher compared to the longest life
hibiscus flowers (3 days). Other cultivars with intermediate flower life showed
intermediate values of ethylene production and ABA content. In fact, analogous results
were observed for ABA content determined in ephemeral and long life flowers. The
concentration in ephemeral flowers was 6-fold (380 ng g-1 FW) higher than in long life
cultivars (63 ng g-1 FW). Petal pigments were also high in petals harvested from the
ephemeral cultivar.
Total carotenoids in ephemeral hibiscus flowers were 30 mg g-1 FW on average
(Fig. 2). Anthocyanins and total phenols were also high in the ephemeral flowers (Table
1). Anthocyanins were 5- to 6-fold higher in the ephemeral flowers (119 mg g-1)
compared to the cultivar with the longest flower life (18 mg g-1). Analogous results were
observed for total phenols with 4-fold difference between the ephemeral (La France)
and the longest life cultivar (Porto).

DISCUSSION
Results obtained confirm that ethylene and ABA are tightly linked with hibiscus
flower life. Ephemeral cultivar with shortest flower life showed the highest hormone
levels, pigments and total phenols. While the longest flower life cultivars had the lower
levels of plant hormones and petal pigments. The high ethylene biosynthesis during
flower life may accelerate flower senescence by autocatalytic induction. In fact,
exogenous ethylene treatments increased ethylene production and shifted earlier the arise
curve of ethylene biosynthesis (Woodson et al., 1985). In the same way low ethylene
production may increase the flower life by delaying all senescence processes. In fact,
ethylene inhibitors such AOA or continuous treatment with 1-MCP extended flower life
(Woodson et al., 1985; Reid et al., 2002; Serek et al., 2006). AOA strongly inhibited
ethylene biosynthesis and delayed flower senescence, demonstrating that ethylene plays
an important role in hibiscus petals senescence (Woodson et al., 1985). Another important
plant hormone involved in flower senescence is ABA, which increases the senescence
processes in many cut flowers (Hunter et al., 2004; Ferrante and Vernieri, 2006). ABA
accumulation showed the same trend as ethylene biosynthesis and endogenous
concentration was highest in short-lived hibiscus flowers. ABA may act by increasing
ethylene sensitivity as observed by exogenous applications of ABA in hibiscus flowers
(Trivellini et al., 2007), suggesting that both hormones are involved in flower senescence.
The ABA biosynthesis may be derived by carotenoids through the indirect biosynthetic
pathway (Milborrow, 2001). Our results showed that the highest carotenoids
concentration was also associated with shortest flower life, suggesting their potential
source for ABA biosynthesis in petals. However, carotenoids degradation and ABA
content should be monitored during flower development and senescence in all cultivars
before to come to a clear conclusion. Anthocyanins were also very high in ephemeral
flowers and they may be linked to senescence processes. In petunias the flower longevity

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was inversely correlated to anthocyanins content (Ferrante et al., 2006). However
anthocyanins are not unusual during flower senescence. In fact, orchid flowers during
senescence change from white to pink or blue (Chadwick et al., 1980). The increase of
total phenols, instead might be due to their antioxidant proprieties and scavengers role
that may play during senescence.
In conclusion, our results suggest that both ethylene and ABA content might be
used as markers for driving the breeding programs that have as aim the improvement of
flower life and persistence on hibiscus potted plants.

ACKNOWLEDGEMENTS
The present work was funded by MIUR with PRIN 2006-2007 Physiological and
molecular aspects of hormonal regulation during the post-production quality of flowering
potted plants.

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Tables

Table 1. Flower longevity, anthocyanins (expressed as cyanindin-3-glucoside) and total

phenols (expressed as acid gallic equivalent) in different cultivars of H. rosa-sinensis
fully open flowers. Values are means with standard errors (n=5).

Cultivars Flower life Anthocyanins Total phenols

(days) (mg g-1 FW) (mg g-1 FW)
Caribbean White eye 2 0.930.199 155.3910.317
Caribbean White 2 0.970.506 159.744.751
Caribbean Tricolor 2 1.480.176 204.2811.314
Caribbean Pink 2 5.130.274 119.7510.222
Caribbean Dark Pink 2 13.271.356 92.754.367
Porto 3 18.401.593 97.204.636
La France 1 119.315.034 431.3523.810

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Figures

Fig. 1. A) Ethylene production from fully open flowers; B) ABA content in fully open
flowers in different cultivars of Hibiscus rosa-sinensis. Values are the means
standard errors (n=3). Data were subjected to one-way ANOVA. Different letters
indicate significant differences (P<0.05).

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Fig. 2. Total carotenoids content in petals of fully open flowers of different hibiscus
cultivars. Values are the means standard errors (n=5). Data were subjected to
one-way ANOVA. Different letters indicate significant differences (P<0.05).

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