Pergamon Phytochem~rry, Vol. 43, No. 2, pp.

3 2 3 - 3 2 6 , 1 9 9 6
Published by Elsevier Science Ltd
Printed in Great Britain
PII: S0031-9422(96)00192-6 0031-9422/96 $15.00 + 0.00

POLYAMINES, ABSCISIC ACID AND ETHYLENE PRODUCTION IN

TOMATO FRUIT

MARIA C. MARTINEZ-MADRID,MARIASERRANO,FERNANDORIQUELME*t and FELIXROMOJARO*

Escuela Politrcnica Superior de Orihuela (UniversidadPolitrcnica de Valencia), Alicante, Spain; *Centro de Edafologla y
Biologia Aplicada del Segura (C.S.I.C.), Murcia, Spain

(Received in revised form 22 February 1996)

Key Word Index--Lycopersicon esculentum; Solanaceae; tomato development and ripening;

slow-ripening cultivar; fast-ripening cultivar; polyamines; abscisic acid; ethylene.

Abstract--Tomato fruit of the fast-ripening cultivar Bretrn had higher rates of ethylene production than the
slow-ripening cultivar Daniela. The initial abscisic acid values were high in both cultivars, but fell during the first
six weeks of development. After this time the levels increased, reaching a maximum before ethylene production
began to increase. This maximum was much more pronounced in the fast-ripening cultivar. The polyamine/
ethylene ratio also differed, with the slow-ripening cultivar having higher levels of putrescine and lower levels of
ethylene. In this cultivar, putrescine was the major polyamine throughout, and it increased sharply during the
ripening stage. Spermidine levels, on the other hand, decreased gradually, especially during the first two weeks of
development. Published by Elsevier Science Ltd

INTRODUCTION intermediate, S-adenosylmethionine (SAM), which is a

substrate for 1-aminocyclopropane-l-carboxylic acid
It is well established that ethylene plays an important
(ACC) synthase in ethylene synthesis and a substrate
role in the ripening process of climacteric fruit. It has
for SAM decarboxylase in a pathway which leads to the
been suggested that other regulators such as abscisic
formation of the polyamines Spd and Spm from Put, by
acid (ABA) and the polyamines, putrescine (Put),
consecutive addition of aminopropyl groups from de-
spermidine (Spd) and spermine (Spm) might also be
carboxylated SAM [2]. In general, ethylene has multi-
important in fruit development and ripening, and at-
ple effects associated with senescence; it initiates fruit
tempts have been made to establish the exact role of
ripening, induces chlorophyll loss in leaves, and pro-
each and their relation with the ethylene action mecha-
motes senescence [7-9]. Polyamines, on the other
nism [1-3]. In non-climacteric fruit, the polyamines
hand, can be considered as senescence inhibitors. They
and ABA might play a much more important role
inhibit the rise in RNase, protease and peroxidase,
because of the lesser importance of ethylene in their
reduce the rate of senescence of leaf protoplasts and
development and ripening.
induce DNA synthesis and mitotic activity [2, 10]. In
The change in concentration of ABA during fruit
avocado, apple, pear and tomato cv Rutgers fruit, free
development differs according to theories. In some
polyamine levels fall during fruit development [ 11 - 15],
cases, ABA concentration increases as the fruit de-
although they increase in mandarin, Shamouti orange,
velops, as in 'Jonagold' apple and avocado [1]. On the
and tomato landrace Alcobaca containing the recessive
other hand in acid lime, sweet cherry and mandarin,
allele alc, during fruit maturation and ripening [16-18].
ABA levels are high during the first stages of develop-
In this work, we study variations in free polyamines,
ment and then fall, remaining low until the fruit ripen
ABA and ethylene, and their possible relationship with
[3-5]. As regards the relationship between ABA and
differences in the ripening patterns of two tomato
ethylene, any interaction there might be is far from
cultivars (cv Bretrn and cv Daniela, fast-ripening and
proved, and there are contradictory views. It has even
slow-ripening, respectively).
been suggested that both hormones act independently
and that ABA has nothing to do with the ripening
process [6].
RESULTSAND DISCUSSION
Both polyamines and ethylene have a common
Fruit growth
A study of the development of the fruit from the two
tAuthor to whom correspondence should be addressed. cultivars led us to divide fruit development into four

323
324 M.C. MARTfNEZ-MADRIDet al.

physiological stages: a) the first two weeks after fruit

set, during which growth occurs through cell division;
b) from two weeks after fruit set until the ninth week,
during which fruits grow through cell expansion in both Daniela /'~
Daniela and Bret6n cultivars; c) the beginning of - • - Bret6n
T
ripening, when the fruit changes from green to red (10
weeks after fruit set); and d) the over-ripening stage, 4
when senescence begins (11 weeks after fruit set in
Bret6n and 12 weeks after fruit set in Daniela).
m 2

Physical-chemical parameters associated with

ripening 2 4 6 8 10 12
In Bret6n tomatoes, firmness and acidity began to Weeks after fruit set
decrease 9 weeks after fruit set and continued to do so Fig. 1. Evolution of ethylene production in Daniela (O) and
until the fruit were totally ripe. These changes began Bret6n (m) tomatoes during development and ripening.
after 10 weeks and were less pronounced in Daniela, Mean_s.e. of the separate measurements made with 20 fruits.
thus explaining its denomination as slow-ripening
(Table 1). The optimum time for picking was 10 weeks
after fruit set for Bret6n tomatoes and 12 weeks for Daniela
Daniela, a time at which firmness and acidity values 250 -l- Bret6n
were similar in both cultivars (Table 1). At 12 weeks,
Daniela tomatoes were just beginning to change colour, ~ 200
while Bret6n tomatoes were already pink. "T
150
o

Ethylene production ~ 100

Both cultivars exhibited a climacteric rise in ethylene 50

production (Fig. 1), which began during the last phases
of fruit development and coincided with the beginning 0 I I I I I I
2 4 6 8 10 12
of ripening. However, it is of interest that maximum
Weeks after fruit set
ethylene production occurred 11 weeks after fruit set in
Bret6n tomatoes with high rates of evolution (8.0 nl Fig. 2. Putrescine levels in Daniela (O) and Bret6n (m)
g - ] hr -~) while in Daniela, ethylene production peaked tomatoes during development and ripening. Meanzs.e. of
between weeks 9 and 11, and with lower values three extractions made in a homogeneous mixture from 20
(maximum around 3.0 nl g - z hr- ). ]
fruits (each extract was quantified in duplicate).

90 nmol g - 1 fr. wt to 40 nmol g - ~ fr. wt. These values

Polyamine levels
remained constant until the 12th week after fruit set
Put, Spd and Spm were present in the pericarp of before falling to 15 nmol g - i fr. wt in both varieties.
both cultivars, although Spin was only just detectable. There was a correlation between Spd levels in tomato
The levels of Put varied between 50 and 100 nmols g - ' mesocarp and the rate of cell division. At fruit set,
fr. wt during the first eight weeks from fruit set. After when cell division occurs, high levels of Spd were
the 10th week these levels fell slightly in Bret6n but found and these levels diminished as the fruit matured
increased 2.5 fold in Daniela (Fig. 2). Spd levels and the rate of cell division decreased, as has been
evolved in a similar way in both cultivars, decreasing found in other fruits, such as avocado, apple and pear
during the first five weeks of development from [11-14].

Table 1. Fruit firmness and titratable activity of Bret6n and Daniela tomato fruits during ripening
Firmness* (kg cm -:) Acidityt (g 100 g-I)
Weeks after
fruit set Bret6n Daniela Bret6n Daniela
8 17.5--_0.4 21.0-+0.6 1.11 -+0.10 0.90-+0.05
9 16.3-+0.5 19.1 -+0.7 0.92-+0.05 0.86-+0.01
10 13.8--+0.3 17.0--+0.5 0.70-+-0.06 0.82-+0.03
11 11.2-+0.2 15.8-+0.3 0.68-+0.03 0.74-+0.03
12 10.0-+0.2 13.9---0.2 0.68---0.02 0.65-+0.01
*Results are given as mean_s.e, of two measurements per fruit for 20 fruits.
tResults are given as mean-+s.e, of the separate measurements made on 20 fruits.
 Polyamines, abscisic acid and ethylene in tomato 325

Daniela shows higher free Put levels and lower in Bret6n tomatoes, in which the ABA content fell by
climacteric ethylene production in ripening fruits than 3.8 nmol g - ~ fr. wt in only two weeks, while it fell by
Bret6n (Figs 1 and 2). The fruits of Daniela also ripen only 1 nmol g-J fr. wt in Daniela over four weeks.
more slowly and keep longer than the fruits of Bret6n We have previously suggested that the effects of free
(Table 1). Since the application of polyamines (Put and polyamines and ethylene are interrelated. Similar con-
Spd) has been shown to inhibit ethylene production in a clusions are reached when the abscisic acid and its
variety of plant tissue [19] including tomato pericarp relation with ethylene production were analysed, In
[18], the high level of free PUt in ripe tissue may be both cultivars, ABA levels increased before the rise in
responsible for the long-keeping quality of Daniela ethylene production took place, although this increase
fruit. High free Put levels have also been observed in in ABA was greater in Bret6n than Daniela (Fig. 3), as
the pericarp of the tomato landrace Alcobaca with the was the increase in ethylene production (Fig. 1). These
recessive allele alc [18] and in the tomato Liberty [15]. results suggest that ABA might act as a promoter of
Both these cuitivars ripen slowly and have prolonged ethylene biosynthesis, the interaction of both hormones
keeping qualities. Furthermore, when inhibitors of stimulating fruit ripening and senescence. This sug-
polyamine biosynthesis were applied to pericarps of gestion is supported by studies of the response of
Liberty, free polyamine levels decreased and ethylene tomato pericarp sections subjected to exogenous ABA
production increased [20]. The high concentration of [21] and studies of fruit development and ripening in
PUt in Daniela may compete for SAM required for sweet cherry, avocado and apple [3].
ethylene production. Also polyamines may directly In conclusion, we think it is safe to suggest that high
inhibit ACC synthase and ACC oxidase which control levels of ABA and ethylene production and low levels
the last two steps in ethylene production. Thus, the of Put are associated with rapid ripening, as occurs in
effects of free polyamines and ethylene are interrelated the climacteric cultivar (cv Bret6n). This hypothesis is
and the high levels of PUt in Daniela cultivar may supported by the comparatively low levels of ABA and
account, at least in part, for the ripening, low ethylene ethylene recorded in the long-keeping cultivar
production rate, and storage characteristics of these (Daniela), in which, furthermore, the concentration of
tomato fruits. Put rose during ripening. The balance between these
three plant development regulators could be responsible
for the different pattern of ripening observed in each
ABA levels
cultivar studied.
The initial ABA concentration was high in both
cultivars (2.5 nmol g-1 fr. wt) falling to 1 nmol g-1 fr.
wt six weeks after fruit set (Fig. 3). Subsequently, in
EXPERIMENTAL
Bret6n, the ABA level increased five-fold during weeks
7 and 8, reaching values of 4.2 nmol g-1 fr. wt and Plant material. Twenty fruits were harvested weekly
then fell sharply (Fig. 3). However, these levels only after fruit set from greenhouse-grown tomato plants
increased two-fold in Daniela between weeks 7 and 9, (Lycopersicon esculentum) cv Bretrn and cv Daniela, in
reaching a value of 1.5 nmol g ~ fr. wt (Fig. 3). These Murcia (Spain). Plants were grown under the same
results point to the marked differences between the conditions of light, temp. and nutrition. The day on
cultivars. The decrease in ABA was extremely marked which fruit set was induced by pollination of flowers by
bumble bees was considered as day 0. The following
determinations were made.
Daniela C2H 4 production rate was measured by placing each
4 --1-- Bret6n fruit in a 0.5-1 glass holder. 1 ml of holder atmosphere
was extracted after 1 hr and the C2H4was quantified
using a GC equipped with flame ionization detector,
3 and a 3-m stainless steel column with an inner diameter
of 3.5mrn containing activated alumina of 80/100
mesh. Ethylene was expressed as C2H 4 given off per g
of tissue per hr (nl g 1 hr- ~).
Fruit firmness was determined with a crosshow
penetrometer (ROZE Paris), fitted with a 3-mm diam-
I L L I I I
eter probe. 2 readings were made on opposite sides of
2 4 6 8 10 12 each of 20 fruits. The results are expressed in kg cm-2
Weeks after fruit set Acidity was determined by potentiometric titration
with 0.1 N NaOH up to pH 8.1 using 1 ml of diluted
Fig. 3. ABA content in Daniela (O) and Bretdn (u) tomatoes
juice in 25 ml H20. The results are expressed as g
during development and ripening. Three extractions were
made from a homogeneous mixture of tissue from 20 fruits. malic acid per 100 g fr. wt.
Four dilutions were made of each extract and each dilution The pericarp of the 20 fruits collected each week was
was quantified in duplicate. Data are mean_+s.e, of these 24 cut into small pieces, to obtain a homogeneous sample,
measurements. and frozen in liquid N 2 before being lyophilized and
326 M.C. MARTfNEZ-MADRIDet al.

stored at - 2 0 ° until the polyamines and ABA were 2. Bagni, N. and Torrigiani, E (1992) in Progress in
analysed. Plant Growth Regulation (Karssen, C. C. M., Van
Polyamine analysis. Since only free, but not conju- Loon, L. and Vreugdenhi, D., eds), p. 264. Kluwer,
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int. standard. The homogenate was then centrifuged for 5. Kojima, K., Kuraishi, S. Sakurai, N. and Fusao, K.
30 min at 20 000 g and free polyamines in the superna- (1993) Sci. Hortic. 56, 23.
tam fr. were analysed by the benzoylation method as 6. Fouad, M., Basiouny, M., Basiouny, K. and
previously described [23]. Derivatives were analysed Maloney, M. (1994) J. Hortic. Sci. 69, 535.
by HPLC. The elution system consisted of MeOH-
7. Mattoo, A. K. and White, W. B. (1991) in The
H20 (16:9) as solvent, run isocratically with a flow
Plant Hormone Ethylene. (Matoo, A. K. and Suttle,
rate of 0.8 ml min -~. The benzoyl-polyamines were
J. C., eds), p. 21. CRC Press, Boca Raton, FL.
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8. Pech, J. C., Balagu6, C., Latch6, A. and Bouzayen,
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M. (1994) Sci. Aliments 14, 3.
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standard and standard curves for Put, Spd and Spm. Mol. Biol. 26, 1579.
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sample and each extraction was quantified by the Physiol. 94, 406.
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quantified as previously reported [24]. The extracts Physiol. Plant. 73, 201.
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containing 1 mM MgC12 and 150raM NaC1, and then chemistry 27, 335.
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