Plant Physiol. (1974) 53, 585-588
Inhibition of Ethylene Evolution in Papaya Pulp Tissue
by Benzyl Isothiocyanate1
Received for publication April 9, 1973 and in revised form December 7, 1973
SURESH S. PATIL AND CHUNG-SHIH TANG
Department of Plant Pathology and Department of Agricultural Biochemistry, University of Hawaii, Honolulu,
Hawaii 96822
ABSTRACT
Papaya (Carica papaya L.) pulp tissue disks in an incubation
medium composed of 0.4 M sucrose evolve ethylene at an optim-um pH of 5.25 at 30 C. Disks of young preclimacteric fruit
evolve the gas linearly with fruit age until fruit age reaches 4
months. Disks from 5-month-old postclimacteric fruit produce
approximately 5-fold more ethylene than disks from 4-monthold fruit. Ethylene evolution by unaged papaya disks is inhibited potently by benzyl isothiocyanate. The compound inhibits production of ethylene by approximately 60% at a
concentration of 0.046 mM. However, in aged papaya disks
benzyl isothiocyanate causes no inhibition of ethylene production indicating that the compound inhibits the induction of the
ethylene-producing system rather than the evolution of the
gas per se. Even at a 2-fold higher concentration benzyl isothiocyanate has no effect on respiration of unaged papaya
disks. It is proposed that benzyl isothiocyanate may act as an
endogenous regulator of ethylene evolution in papaya fruit.
papaya fruit release traces of BITC2 vapor. In the course of a
study (10) involving the role of BITC in the natural defense
of papaya against fungal pathogens, we discovered that fruit
treated externally with BITC failed to degreen normally. On
the assumption that ethylene acts as a ripening hormone in
papaya also, we reasoned that the failure of BITC-treated fruit
to degreen normally might be due to BITC interference with
the evolution or the action of ethylene. Here we report that
papaya pulp tissue evolves ethylene and that BITC inhibits
such evolution.
MATERIALS AND METHODS
Chemicals. All chemicals were reagent grade. BITC was obtained from K and K Laboratories Inc. An aliquot of BITC
was washed by vigorously shaking with 50 volumes of deionized water in a separatory funnel. After repeating the process three more times, the washed sample was used to make the
saturated standard stock solution. This solution, which contained 0.92 ,umole ml of BITC, was used in the experiments
reported here. As a check, a gas chromatographically pure
sample of BITC was employed in one experiment. The effects
of both the water washed and GLC purified BITC samples on
ethylene evolution of papaya disks were comparable.
Gas Analysis. Gas samples were analyzed essentially acIt is now well established that in detached maturing fruit of cording to Meigh et al. (6) with the use of an alumina column
many plant species ethylene acts as a ripening hormone. How- (50 c) on a Varian gas chromatograph equipped with a flame
ever, little is known regarding the regulation of ethylene pro- ionization detector. Unless otherwise stated, 1 cc gas samples
duction in detached fruit. Auxins and cytokinins (1) and were used for gas analysis.
Papaya fruit. Papaya (Carica papaya L.) fruit of the Solo
gibberellin (4) are all implicated directly or indirectly in the
stimulation of ethylene evolution in various plant tissues. The variety were obtained from the Puna District of the Island of
existence of specific, naturally occurring compounds, which Hawaii. All the experiments except one were done with pulp
seem to either inhibit ethylene production or to reduce the sen- disks taken from postclimacteric fruit which were approxisitivity of tissues to ethylene, has been suggested by Meigh et al. mately 5 months old and still green on the surface when de(7) and Burg and Burg (3). Sakai and Imaseki (11) observed a tached from the tree. In one experiment, the ability of pulp
rapid decrease in the rate of auxin-induced ethylene produc- tissue to evolve ethylene was measured on tissue taken from
tion of mung bean hypocotyls upon the removal of auxin from immature fruit of various ages. Fruit for this experiment was
or the addition of cyclohexamide to the incubating medium. collected on the basis of size which was used to estimate the
To explain the data they suggested that the auxin-induced eth- age of the fruit. The age of preclimacteric fruit used was 1, 2,
ylene-producing system is rapidly degraded (turned over) by 3, and 4 months. Fruit was stored at 10 C and used within
4 to 5 days after it was detached.
another system in vivo.
Ethylene Studies. Papaya plugs (12 mm in diameter) taken
Maturing whole papaya fruit evolve ethylene and application of the gas initiates a respiratory rise and ripening in de- with a cork borer were hand-sliced into 3-mm thick discs with
tached fruit (2). Papayas ubiquitously contain benzyl glucosi- a razor blade. The first disk which included the epidermis was
nolate. Injury to the fruit induces enzymatic hydrolysis of the discarded and only the second disk from each plug was used
glucosinolate and release of benzyl isothiocyanate (12), a com- for ethylene studies. Disks were standardized on the basis of
pound with antifungal properties (13). Even uninjured intact color and those ranging from greenish white to faintly yellow
were selected for use. Five preweighed disks were suspended
in 5 ml of the appropriate incubating solution in a 50-ml Er1 Hawaii Agricultural Experiment Station Journal Series No.
'Abbreviation: BITC: benzyl isothiocyanate.
1603.
585
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586
PATIL AND TANG
lenmeyer flask. The flask assembly consisted of the Erlenmeyer
flask, a neoprene stopper and glass tube, and a gas tight
septum. The gas volume of each assembly was calculated individually.
The contents of the flasks were chilled until the start of the
experiment. At appropriate timed intervals, flasks were affixed to Warburg manometers with rubber bands, lowered into
a water bath (30 C), and were shaken at 100 strokes per min.
In order to maintain the pressure in the flasks to atmospheric
level, a volume of air equal to the volume to be withdrawn was
injected into each flask just prior to withdrawal of the gas sample.
The effect of BITC on ethylene production of papaya disks
was studied in two ways. In one, appropriate amounts of BITC
were added to flasks which contained unaged papaya disks in
the incubation medium (0.4 M sucose). In the other, disks were
preincubated (aged) for 3 hr in the incubating medium; washed
with fresh incubating medium and then transferred to flasks
which had the medium containing appropriate amounts of
BITC.
For determining the pH optimum of ethylene evolution,
papaya disks were incubated in 5 ml of 0.4 M sucrose made
with buffers of appropriate pH values; 0.1 M Na citrate-phosphate was used to obtaining buffers of pH values of 3, 4, 5, and
6. Other buffers used for studying roughly the same pH range
were 0.1 M Na citrate-citric acid, KHphthalate-NaOH, and
0.05 M K2HPO4-citric acid. The effect of BITC on respiration of
papaya disks was studied by determining the rate of CO2 evolution. The experimental conditions used here were the same
as those described for the BITC-ethylene experiments, except
that the diameter of pulp disks was 7 mm and the volume of
the incubating solution was 4 ml. Evolution of CO2 in the
presence and in the absence of BITC was measured with a
Warburg respirometer at 30 C.
RESULTS
Plant Physiol. Vol. 53, 1974
5
3:
{- 4
Cn
0
L-
3
'
2
-.
0 1
En
0
0
EC
I
6
5
pH
FIG. 2. Dependence of ethylene evolution of papaya disks on
the pH of the incubation medium. The medium was prepared by
dissolving sucrose in 0.1 M citrate-phosphate buffer of appropriate
pH values. Samples were taken after rate of ethylene evolution
became linear with time (between 3 and 4 hrs after incubation
began).
3
4
40
.C
IC)
c
0.
30
20
CL
Figure 1 shows the time course of ethylene production by
unaged papaya disks. A lag period of 2 to 3 hr occurs before
ethylene evolution becomes linear with time. Detectable quan-
Ec
10
1
2
'C 15
3
4
5
Age (months)
FIG. 3. Relationship between ethylene evolution of papaya
disks and the age of the parent fruit. All conditions of the experiment were as in Figure 1, except that each point represents a
separate experiment with five replicate flasks. Samples were taken
after a 5-hr incubation period.
10
C
5
tities of the gas are present at 20 min after the start of the experiment. Maximum ethylene evolution occurs at about pH
0
5.25 (Fig. 2) in 0.4 M sucrose made in 0.1 M citrate-phosphate
E
buffer. The same pH optimum was observed when the 0.4 M
C
I
sucrose was made with acetate, phthalate, or citrate-phosphate
2
3
4
5
buffers. However, in 0.4 M sucrose solution made with deionized water, the ethylene evolution by papaya disks is identical
Time (hr)
to that produced in the buffered sucrose solution of optimum
FIG. 1. Time course of ethylene evolution in papaya pulp disks.
except for the pH study, all experiments were
Five unaged disks, from postclimacteric papayas, weighing ap- pH. Therefore,
M
0.4
done
sucrose made with deionized water as the inusing
M
2
ml
of 0.4 sucrose solution
proximately g were suspended in 5
medium.
cubating
in the Erlenmeyer flask and incubated at 30 C as described under
The production of ethylene by disks taken from fruits at
"Materials and Methods." One cc of air was injected in the flask
just before withdrawing the sample of the same volume. Gas various stages of maturity is shown in Figure 3. Ethylene
evolution increases linearly with the age of the fruit up to 4
analysis was done according to Meigh et al. (5).
0
1
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Plant Physiol. Vol. 53, 1974
587
ETHYLENE INHIBITION BY BENZYL ISOTHIOCYANATE
strongly reduces ethylene evolution of papaya tissue. However,
this could be an indirect result of a general inhibition of some
other important process such as respiration. We studied the
effect of BITC on the respiration of papaya pulp disks. Data
in Figure 6 show that there is no appreciable inhibition in the
CO2 evolution by BITC at concentrations that inhibit ethylene evolution.
40
30
DISCUSSION
-0-
0--
in
4)
C0 L.
20
The results presented here show that papaya pulp tissue is
capable of producing ethylene. The use of pulp disks instead
of whole fruit facilitates further studies on the role and biogenesis of ethylene in papaya tissues. Although little detailed
work on the role of ethylene in papaya ripening has been reported, Akamine and Goo (2) have observed accelerated ripening in fruit externally treated with ethylene gas. In this study
when ethylene gas (30 p.l in 0.5 ml air) was injected in the
central cavity of postclimacteric papaya, they ripened much
faster than noninjected fruit (Patil, unpublished). Therefore,
DI
Ec
10
5
Time (hr.)
3
4
6
FIG. 4. Inhibition of ethylene production by BITC. Appropriate
amounts of a saturated solution (0.92 ,umole/ml) of BITC were
incorporated in the incubation medium. The concentrations were:
0.023 mM (A); 0.046 mM (B); 0.092 mm (C); and 0. 184 mm (D).
Unaged papaya disks were used.
100
D~~~
80
Table 1. Effect oJ BITC oni Ethylene Evolutioni of Aged Papaya
Pulp Tissue
Five disks from postclimacteric fruit were aged in 5 ml of incubation medium (0.4 M sucrose) for 3 hr, washed with the same
medium, and resuspended in 5-ml portions of the incubation
medium which contained appropriate concentrations of BITC.
The rate of ethylene evolution between 2nd and 3rd hr (after exposure to BITC) was determined. For control, the rate was 14.3
nmoles/g fresh wt-hr.
BITC
o 60
"I
0.023
0.045
0.09
0.180
C 40
20
Ethylene Evolution
%go of control
94
94
92
97
3
00
0.05
0.1
0.15
0.2
BITC (mM)
FIG. 5. BITC concentration versus inhibition of ethylene evolution. The dosage-response curve was plotted by using ethylene
evolution data obtained at 4.5 hr in the experiment described in
Figure 4.
months, but disks from 5-month-old (postclimacteric) fruit
produce approximately 5-fold more ethylene than disks of
fruit 4 months old.
The effect of four concentrations of BITC on the time course
of ethylene production by unaged papaya disks is shown in
Figure 4. The same data are presented in the form of a dosage
response curve (Fig. 5), except that only one reading for gas
evolution (4.5 hr) was used for plotting the graph. Inhibition
of ethylene evolution is linear with the concentration of BITC
only up to approximately 60%. Further increase in the concentration of the inhibitor results in only a slight increase in
the inhibition of ethylene production. When the same experiment is repeated with disks which were aged for 3 hr, no correlation between BITC concentration and per cent inhibition
was observed (Table I), although there was some inhibition of
ethylene production at all four concentrations of BITC tested.
It is evident from the data in Figures 4 and 5 that BITC
o Control
3.
*A
4)
0
_.
aB
A C
t-2
0
x
1
0
u
1
2
3
4
Time (hr.)
FIG. 6. Effect of BITC on CO2 evolution of unaged papaya
disks. BITC concentrations were: 0.023 mM (A); 0.046 mM (B);
and 0.092 mM (C). Papaya disks (7 mm in diameter) were incubated in 4 ml of 0.4 M sucrose solution in Warburg flasks. CO2
evolution was measured at 30 C according to standard manometric
techniques.
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588
PATIL AND TANG
it appears that ethylene plays the same role in papaya as it
does in other climacteric fruit.
The inhibition of ethylene production by BITC in unaged
but not in aged papaya tissue indicates th-at the effect of the
inhibitor is on the induction of the ethylene-producing system
rather than on the evolution of the gas after the system is induced. The mechanism by which BITC inhibits this induction
is not known. However, the lack of inhibitory effect of the
compound on respiration of unaged papaya tissue suggests
that it has a degree of selectivity with regard to inhibition of
processes (such as protein synthesis) involved in tissue aging.
It is possible that BITC suppresses synthesis of enzymes involved in the biosynthesis of, for instance, the ethylene precursor methionine.
The data on the effect that BITC treatment on ethylene evolution of papaya disks support our hypothesis (10) that BITC
interferes with the normal ripening of papaya by inhibiting
their ethylene evolution. It is not possible to determine accurately the endogenous concentrations of BITC in papaya
because maceration of tissue readily hydrolyzes the benzyl
glucosinolate to BITC. However, concentrations of glucosinolate in papaya tissue as high as 2 ,umoles/g fresh wt have
been calculated (12). If only a small percentage of this total
glucosinolate existed in the form of BITC it could qualify for
the role of an endogenous regulator of ethylene evolution in
papaya tissues. The latter view is further reenforced by the
following: on the one hand, there exists an inverse correlation
between age of papayas and the BITC concentration in the
pulp (12); and on the other hand, as current results show,
there is positive correlation between the age of the fruit and
the ability of the pulp disks to evolve ethylene. Thus, there appears to be a cause-effect relationship between high BITC
concentrations in young fruit and the low capacity for ethylene evolution by disks taken from such fruit.
There are very few reports of specific inhibitors of ethylene biogenesis (5). Owens et al. (9) reported that Rhizobitoxin,
a bacterial peptide toxin, is a potent inhibitor of ethylene
Plant Physiol. Vol. 53, 1974
evolution in sorghum seedlings and apple pulp tissue. The
compound has been reported (8) to inhibit /1-cystathionase of
Salmonella typhimurium and of spinach. The inhibition of
ethylene appears to be due to nonconversion of methionine
to ethylene rather than the inhibition of methionine synthesis
itself. Also, there are reports (4, 7) which suggest the existence
of naturally occurring inhibitors of unknown identity which
act as endogenous inhibitors of ethylene production in attached fruits of two plant species. To our knowledge this is
the first report describing an identified endogenous inhibitor
of ethylene evolution in plant tissues.
LITERATURE CITED
1. ATiELES, F. B. AND B. RUBENSTEIN. 1964. Regliatioii etlf
etyl(n evolution
an(i leaf abscission by auxin. Plant Physiol 39: 963-969.
2. AKAMINE, E. K. AND T. Goo. 1973. Respiration, ethylene production, antl
shelf-like extension in irradiated papaya fruit after storage under simullatedl
shipping cond(lition. Haw. Agri. Exp.Sta. Tech. Bull. No. 93. In press.
3. BuR;., S. P. ANi) E. A. B-ac. 1965. Ethylene action an(d the ripening of
flrtits. Scienice 148: 1190-1196.
4. LIBERMAN, M. AND L. W. MAPSON. 1964. Genesis aud(t biogenesis of ethylene.
Naturle 204: 343-345.
5. McGLASSON, W. B. 1970. The ethylene factor. In: A. C. Hiilme, ed., The Biochemistry of Frtuits an(d Their Pro(duiets. Vol. 1. Acadeimiic Press, New York.
pp. 475-520.
6. 'MEIGH, D. F., K. H. NoRiRis, C. C. CRZAFT, AND M. Luu:iiauN. 1960. Ethlylene
production by tomato and apple fruit. Nature 186: 902-903.
7. MEIGH, D. F., J. D. JONES, AND A. C. HULSIE. 1967. Thie respiration
climactelic in the apple. Phytocliemistry 6: 1507--151,5.
8. OWENS, L. D., S. Gt-GGENHEIM, AND J. HILTON. 1968. I)ilzobitinu1-syntliesized
phytotoxin: an inhibitor of /3-cystathionase in Sa.Inionella typhinmurini.
Biochiim. Biophys. Acta 158: 219-225.
9. OWE:NS, L. D., M. LIEBERMAN, AND A. KuNi.SHI. 1972. Iibiiitition of ethylene
pro(dtuction by Phizobitoxin. Plant Physiol. 48: 1-4.
10. PATIL, S. S., C. S. TANG, AND J. E. HUNTER. 1973. Effect of benzyl isothiocyanate treatrueiit on the development of postliarvest rots in papayas. Plant
Dis. Rep. 57: 86--89.
11. SAKAI, S. AND) HI. IMASEKI. 1971. Auxin-induce(d ethylene production by
mung bean hiypocotyl segnments. Plant Cell Physiol. 12: 349-359.
12. TANG. C.-S. 1970. Benzyl isothiocyanate of papaya frujit. Phytochiemistry
10: 117-121.
13. VIRTANEN, A. 1. 1965. Stu(dies on organic sulfur compounds aid(i tither labile
suhstances in plilats. Phivtochemistry 4: 207-228.
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