Postharvest

Biologyand
Techr~locjy
ELSEVIER Postharvest Biologyand Technology4 (1994) 167-177

Effect of ethylene treatment on ethylene production,

EFE activity and ACC levels in peel and pulp
of banana fruit
M. D o m i n g u e z , M. V e n d r e l l *
csIc, Centro de lnvestigaci6n y DesarroUo,Jorge Girona 18-26, 08034 Barcelona, Spain
(Accepted 21 September1993)

Abstract

Exogenous ethylene treatment (100 ppm) induces preclimacteric bananas (Musa acumi-
nata Collar. cv. Dwarf Cavendish) to ripen with increased respiration and endogenous
ethylene production; 12 h treatment was slightly more effective than 6 h. In transverse
sections of bananas the contribution of the peel to respiratory and ethylene increases af-
ter induction by exogenous ethylene was negligible and the pulp provided all the output.
Increases in respiratory CO2 and ethylene by banana slices were generally in line with the
inductive period of ethylene treatment. Banana slices treated with ethylene show an increase
in EFE activity in both peel and pulp, with that in the peel being shown earlier. The effect
is related to the duration of treatment. Ethylene-treated slices contained higher levels of
conjugated ACC, but not free ACC, than control slices, but there was no clear link with
the length of exposure to exogenous ethylene. The increase in ACC conjugation activity
could explain the initial lower ethylene production shown by slices treated with exogenous
ethylene.

Key words: Bananas; Banana peel; Banana pulp; Ethylene induction; ACC; EFE activity

1. Introduction

In plant tissues, ethylene is produced from methionine via S-adenosyl methion-

ine (SAM) and 1-aminocyclopropane-l-carboxylic acid (ACC) (Yang, 1981). ACC
synthase and ethylene forming enzyme (EFE) are the key enzymes in the control
of ethylene biosynthesis (McGlasson, 1985). In climacteric fruits, regulation of ethy-
lene biosynthesis seems to depend as much on ACC availability as on the tissue
capacity to convert ACC to ethylene (Yang, 1987). The ACC level can be regulated

* Corresponding author. Fax: 34 (3) 204 59 04.

0925-5214/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved.

SSDIO925-5214(93)EO055-I
168 M. Domlnguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177

by the rate of its synthesis and its conversion to ethylene and by its conjugation to
malonyl-ACC (MACC) (Yang and Hoffman, 1984). On the other hand, ethylene is
capable of regulating the activity of EFE (Hoffman and Yang, 1982), ACC synthase
(Buffer, 1984) and malonyl-ACC transferase (Liu et al., 1985b).
Treatment of climacteric fruits with ethylene induces ripening (Burg and Burg,
1962). Concentrations as low as 0.1 ppm ethylene can be effective in preclimacteric
bananas (Peacock, 1972), although commercially treatments of about 1000 ppm are
used (Inaba and Nakamura, 1988). The effect of any treatment depends on the
sensitivity of the fruit, which increases with age, on ethylene concentration and
on the period of exposure to ethylene (Inaba and Nakamura, 1986). The effect of
ethylene seems to be related to its capacity to combine to receptors present in the
tissue (Whitehead and Boss6, 1991).
In this paper, we show the effect of different periods of exogenous ethylene
treatment on ethylene biosynthesis in whole banana fruit, transverse sections and
pulp and peel tissue.

2. Material and methods

Plant material
Bananas (Musa acuminata Collar. cv. Dwarf Cavendish), slightly immature by
commercial standards, were obtained from Tenerife. Fruits from the same hand
(15-20) were used in each experiment to avoid differences in physiological develop-
ment.
The bananas were washed with an aqueous fungicide solution containing 0.1%
(w/v) Benlate and 0.2% (w/v) Ditane, and allowed to dry. Single fruits were placed
in respiration jars and ventilated with humidified air (flow about 1.2 1 h -1) at 24°C.
In each experiment at least three whole fruits were kept as controls, measuring
respiration and ethylene production.
Transverse 6 mm-thick whole slices were cut from whole fruits and handled as
described by Palmer and McGlasson (1969). Sections of pulp or peel tissue were
prepared by removing the peel from 6 mm-thick slices (Vendrell and McGlasson,
1971).

Ethylene treatment
Individual whole fruits or composite samples of slices (about 30 g FW) or of peel
or pulp tissue were used. Fruit sections were treated 24 h after cutting. This is the
time they take to recover from the mechanical stress of cutting. Samples were placed
in closed 2-1 jars, with a septum, through which ethylene (from a mixture of 5%
in nitrogen) was injected to create a concentration of 100 ppm, which was verified
by gas chromatography. Inside the jars a 20% solution KOH was placed in a small
container to absorb the CO2 produced, as described by Liu et al. (1985a). After
treatment (6, 12, or 24 h), samples were ventilated to eliminate the ethylene and
placed in ventilated jars at 24°C. Endogenous ethylene production and respiration
were measured (see below). Some samples were used immediately after treatment
to analyze EFE activity or ACC and MACC content.
 M. Dominguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177 169

Analysis of ethylene production

Single fruit or composite samples of slices were placed in ventilated jars and
2-ml samples were taken from the effluent air from each respiration jar and injected
into a Perkin-Elmer 3920B gas chromatograph with a gas-tight syringe. Three fruit
samples were used per treatment. The gas chromatograph was fitted with a flame
ionization detector. Operating conditions were as follows: the column (2 m x 4
mm i.d.) was packed with 80-100 mesh activated alumina, oven temperature ll0°C,
nitrogen carrier gas 40 ml rain -1 .

EFE activity
EFE activity was measured in vivo as described by Mansour et al. (1986). Peel
and pulp discs, one of each, from three transverse whole slices, about 1 mm thick
were incubated in the presence or absence of 5 mM ACC. Ethylene production was
measured by gas chromatography after three hours of incubation at 24°C. Results
are given in nmol C2H4 gFW -1 h -I.

ACC and MACC assay

ACC and MACC were measured in peel and pulp. In each measurement, tissue
samples were taken from three transverse whole slices and placed immediately on
dry ice. Samples of 1-2 g were extracted with 80% ethanol under reflux for 15 min.
Ethanol was removed under vacuum at 40°C. Free ACC was measured directly on
the aqueous extract following the method of Lizada and Yang (1979). Total ACC
(free and conjugated) was measured from the aqueous extract hydrolysed with HC1
(2N) at 100°C for 3 h, following the method of Hoffman et al. (1982). Results are
given in nmol gFW -1.

Analysis of respiration
Respiration was measured as COz production. The effluent air from the venti-
lated jars was connected to a Cossma infrared gas analyzer, model Diamant 6000.
Results from three fruit samples are given in/zg CO2 gFW -1 h -1.

Statistical analysis
ANOVA and two-way ANOVA were used to compare treatments. Differences
between treatments are considered at a P < 0.05 level of significance.

3. Results

Effect of ethylene treatment on respiration and ethyleneproduction in whole fruits

Fig. 1 shows the effect of treatment with 100 ppm ethylene for 6 or 12 h on
respiration and ethylene production by whole bananas. Treatment with ethylene
induces an increase in respiration similar to the respiratory climacteric. The rise in
CO2 production is immediate and related to the time of treatment (Fig. 1A). In
the 12-h treatment the climacteric peak is reached in 3 days. The 6-h treatment has
a similar effect but the climacteric peak is attained 1 day later. In both cases the
170 M. Domfnguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-I 77

150

0 ~) I I I I
5 10 15 20
B

~- 3
"7

0 ,-_ i I
"" I
0 10 15 20
Days after treatment
Fig. 1. Effect of ethylene treatment (100 ppm) on respiration rate (A) and ethylene production (B)
in whole bananas treated for 6 (zx) or 12 (A) h compared to controls untreated (0). Three fruits
were used per treatment. Statistical analysis (ANOVA) shows significant differences between both
treatments and controls, and between the two treatments (P < 0.001).

treated fruits show a slightly higher maximum of respiration (150/~g CO2 gFW -1
h -1) than untreated fruits (130 /zg CO2 gFW -1 h - l ) , probably due to a higher
synchronization in the induced respiration in the fruit tissue.
The effect of ethylene treatment on endogenous ethylene production shows
similar results to those for respiration (Fig. 1B). The 12-h treatment induces an
immediate increase in ethylene production, which reaches a maximum in 2 or 3
days (1.5 nl gFW -1 h - l ) , although this level is lower than that attained by control
fruits (3-3.5 nl gFW -1 h - l ) . However, the ethylene production in treated fruits then
increases steadily to reach the level of the controls, but showing clear symptoms of
senescence (browning and softening). The 6-h treatment shows similar results than
the 12-h, but with about i or 2 days delay.
 M. Dominguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177 171

Effect of ethylene treatment on respiration and ethylene production by transverse whole

slices
Fig. 2 shows the effect of treatment with 100 ppm ethylene for 6, 12 or 24 h on
whole slices, on respiration and on ethylene production. Treatment was applied 24 h
after cutting, when the slices had already recovered from the mechanical stress. A
treatment of 6 h induces an increase in respiration (100-110/zg CO2 gFW -1 h -1)
(Fig. 2A) that then decreases to lower levels, but higher than the controls. These
samples show an advance of the climacteric rise of two days compared to controls. A
treatment of 12 h induces a higher increase in respiration than the 6-h treatment, to
the level of 130-140/xg CO2 gFW -1 h -1 which, after a slight decrease, again returns
to the levels of the climacteric peak. A treatment of 24 h has an even stronger effect
on respiration.

A
;~: 150
7"
u.

0"100
¢..)

50
I I I
() 5 10 15
B
8
i

~= 6
14.
O~
4

"E
2

0 5 10 l's
Days after treatment
Fig. 2. Effect of the length of exposure to exogenous ethylene (100 ppm) on respiration rate (A) and
ethylene production (B) of whole transverse banana slices. Treatment was applied 24 h after cutting,
for 6 (O), 12 (zx) or 24 (t3) h and are compared to controls untreated (e). Three composite samples
were used per treatment. In all eases, there are significant differences with the controls ( P < 0.05,
P < 0.001 and P < 0.001, respectively).
172 M. Domtnguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177

The endogenous ethylene production of whole slices treated for 6 h (Fig. 2B) is
similar to controls except that the increase to a peak characteristic of the climacteric
is advanced 2 days, as the respiration increases. In whole slices treated for 12 or 24
h, ethylene production is not parallel to the increase on respiration. Immediately
after treatment the production levels are low, and they increase steadily after about
5 days, but in a different pattern from that of normal ripening in controls.

Effect of ethylene treatment on respiration and ethyleneproduction in peel and pulp

sections
The effect of treatment with 100 ppm ethylene for 12 h on peel and pulp
sections, obtained from the same fruits and treated simultaneously, is shown in Fig.
3. The pulp shows a quick rise in respiration that reaches the level of the normal
climacteric peak in banana, and higher (Fig. 3A). However, in the peel there is an

250 A p- - 0 " ~ " ° " 0

"T

o~ 150 y P " o - - d / ° " ° ' ~

,="50
o ,'o 1'5
. 8 r-
B

%
• .JC)
;-" 4
"2

0
0 5 10 15
Days after treatment
Fig. 3. Effect of ethylene treatment (100 ppm for 12 h) on respiration rate (A) and ethylene production
(B) in peel (zx) or pulp (©) sections, obtained from banana fruit, compared to controls untreated (A)
peel and (e) pulp. Three composite samples were used per treatment.
 M. Dominguez, M. l,'endrell/ Postharvest Biology and Technology 4 (1994) 167-177 173

initial increase in respiration, but afterwards it decreases to the low levels prior to
treatment, and similar to that of control untreated peel.
The pattern of ethylene production is somehow similar (Fig. 3B). The peel does
not show any effect. The pulp shows an increase in endogenous ethylene production
but the pattern is different from that of controls. There is an initial increase (3.5-4
nl gFW -1 h -1) but not to the level of the climacteric ethylene peak of pulp controls
(8 nl gFW -1 h-]); production then decreases, and then increases again.

Effect on EFE activity of peel and pulp of transverse whole slices

EFE activity, in peel and pulp of whole banana slices treated with ethylene (100
ppm) 24 h after cutting, was measured just after the different periods of treatment
(3, 6, 12 or 24 h) (Fig. 4). There is an increase in EFE activity, in both peel and
pulp, in treated slices compared to controls. This increase, in the peel (Fig. 4A), is
evident after a 6-h treatment. In the pulp (Fig. 4B), where EFE activity is lower,
the differences are not clearly discernible before a 12-h treatment. In both, peel and
pulp, the differences compared to controls become larger in longer treatments.

2.0

1.0

e-
0
6 ll2 24
B
0.4

0.2

C
0 . i
o 8 12 2'4
Hours of treatment
Fig. 4. EFE activity in peel (A) and pulp (B) of whole banana slices treated with ethylene (100 ppm)
24 h after cutting, for different periods (B) compared with controls untreated (e). EFE activity was
measured just after each period of treatment. Three composite samples were used per treatment. All
results show a significant difference compared to controls ( P < 0.05).
174 M. Dominguez, M. Vendrell/ PostharvestBiologyand Technology4 (1994) 167-177

~ 4

'7
3
B

__J J J"
6 2

" 1

0 t
o 12 2'4
Hours of treatment
Fig. 5. Free (n) and total ACC (free + conjugated) (a) content in peel (A) and pulp (B) of whole
banana slices treated with ethylene 24 h after cutting, for different periods compared with controls (O,
free ACC and e, total ACC) untreated. ACC was measured just after each period of treatment. Three
samples were used per measurement. Fruit samples are the same as for Fig. 4. Changes in free plus
conjugated ACC in both peel and pulp, and of free ACC in peel are significant compared to controls
(P < 0.05), but not in free ACC in pulp (P < 0.1).

Effect on free and conjugated A C C in peel and pulp of transverse whole slices
Ethylene treatment (100 ppm) increases or prevents a decline in total A C C
(free + conjugated) both in peel and pulp (Fig. 5). As in the m e a s u r e m e n t of
E F E activity, the longer the treatment the differences compared to controls are
slightly higher, but the link of the level of total A C C with the length of exposure to
exogenous ethylene is not clear. However, free A C C in the pulp of the slices (Fig.
5B) does not differ significantly from that of the controls. In peel (Fig. 5A), free
A C C in treated slices is slightly higher after a 12-h treatment.

4. Discussion

The use of 100 p p m ethylene for 24 or 48 h to induce ripening of bananas at

a t e m p e r a t u r e between 15 and 25°C is a c o m m o n commercial practice (Scriven et
al., 1989). Several authors have used concentrations ranging from 1 to 1000 p p m
(Vendrell and McGlasson, 1971; Inaba and Nakamura, 1988; Ke and Tsai, 1988).
 M. Dorninguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177 175

From the results obtained after treatment of whole bananas with 100 ppm for 6 or
12 h, it can be deduced that endogenous ethylene production does not follow the
same pattern as in natural ripening. The characteristic ethylene climacteric peak
does not appear, and the level of ethylene production is lower, although increasing.
Natural ripening and ethylene production seems to begin in the pulp, and ripening
of the peel depends on the pulp (Vendrell and McGlasson, 1971; Garcia and Lajolo,
1988; Dominguez and Vendrell, 1993). There are differences in flavour and aroma
in bananas ripened with exogenous ethylene compared to natural ripening (Scriven
et al., 1989). Besides, inhibition of endogenous ethylene production caused by
exogenous ethylene treatment (Vendrell and McGlasson, 1971), could be the cause
of the different behaviour of treated fruits. The results obtained are similar to those
described by Ke and Tsai (1988) and by Whitehead and Boss6 (1991).
Transversal banana slices show a ripening behaviour similar to that of whole
fruits. They exhibit the characteristic climacteric peak of respiration, autocatalytic
ethylene production, chlorophyll breakdown in the peel and starch hydrolysis in
the pulp (Palmer and McGlasson, 1969; Vendrell and McGlasson, 1971). The
behaviour of whole slices treated with ethylene shows the need of a minimum
treatment period to induce the climacteric respiration rise, although not initially
the autocatalytic ethylene production probably due to the inhibition of endogenous
ethylene production caused by ethylene treatment. With 100 ppm, the minimum
treatment period is between 6 and 12 h. Shorter periods do not induce the
climacteric, but make the tissue more sensitive to ethylene (Halevy and Mayak,
1981) and the onset of ripening is advanced. According to Yang (1987), the effect
of exogenous ethylene could be on receptors of system I, also inducing the increase
of respiration and disinhibiting the receptors of system II or their synthesis. In any
case, sufficient effect to produce ripening is only obtained over time.
The data of Fig. 3 indicate that the peel is incapable of autocatalytic ethylene
production. It does not respond to ethylene treatment unless this is continuously
present and even then, changes are at the level of pigment development (Ke
and Tsai, 1988). This is probably because the peel does not have the capacity of
system II for autocatalytic ethylene production, but only that of system I, which
produces lower amounts of ethylene related to senescence. However, the pulp
responds irreversibly to ethylene treatment. Differences with controls in the pattern
of ethylene production in ethylene-treated pulp slices could be due to the initial
inhibition caused by ethylene treatment (Vendrell and McGlasson, 1971).
The effect of ethylene on EFE is to enhance enzyme activity. Enzyme activity
is higher in the peel by about a factor of 5. Similar results have been obtained in
tomatoes, melons (Liu et al., 1985a) and in apples (Buffer, 1986).
As total ACC is higher in slices treated with ethylene, while free ACC does not
change (in the pulp) it follows that the MACC level increases. Then exogenous
ethylene may increase ACC synthase activity (Buffer, 1984), but at the same time
as, and faster than, the malonyl-ACC transferase activity (Liu et al., 1985b). ACC
produced is converted almost immediately to MACC, increasing MACC level. If
ACC does not accumulate, it has no access to EFE, whose activity is increased
by exogenous ethylene, and is not metabolized to ethylene. This could explain the
176 M. Domtnguez, M. Vendrell/ Postharvest Biology and Technology 4 (1994) 167-177

initial inhibition of endogenous ethylene production observed in bananas treated

with ethylene (Vendrell and McGlasson, 1971), and it is probably related to the
low ethylene production rates observed later in banana slices treated with ethylene
(Figs. 2B and 3B).

Acknowledgements

The authors are grateful to Dr. J. Hermindez from Dpto. Protecci6n Vege-
tal, C.I.T.A., Tenerife, Canary Islands, for the supply of bananas. This work was
supported by grants ALI-88-0138 and ALI-91-1122-C03-01 from Comisi6n Inter-
ministerial de Ciencia y Tecnologia.

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