Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue3-4.June2000:
Scientia Horticulturae 84 (2000) 309±319
Effects of auxin on growth and ripening
of mesocarp discs of peach fruit
Akemi Ohmiya*
National Institute of Fruit Tree Science, Ministry of Agriculture,
Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan
Accepted 8 November 1999
Abstract
A bio-assay system using the mesocarp discs of peach fruit (Prunus persica L. cv. Akatsuki) was
developed, and the effects of auxin on the physiology of fruit tissues were investigated at different
stages of development (FWI: initial period of exponential growth, FWII: period of slow growth, and
FWIII: second period of exponential growth). Auxin promoted both the enlargement of discs as well
as ripening processes such as softening and anthocyanin formation. In particular, discs at FWI and
FWII enlarged remarkably after four weeks of incubation with NAA. The highest value of the
weight of FWI discs was observed at 10 mM of NAA; that of FWII discs was at 1 mM. Weights were
compared between discs that were incubated with and without NAA. NAA-incubated discs reached
to 3.1-fold in the FWI stage and 2.7-fold in the FWII stage. Discs at each stage of development lost
their ®rmness with increasing concentrations of NAA up to 100 mM. There was a signi®cant
difference in anthocyanin formation between light- and dark-incubated discs. Anthocyanin
formation at the surface of the discs was enhanced by high concentrations of NAA and light,
whereas that inside of the discs was enhanced by darkness and low concentrations of NAA. These
multiple effects of auxin on growth and the ripening process of fruit tissue may be caused by
multiple mechanisms of auxin action as in¯uenced by the stage of fruit tissue growth and
environmental conditions. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Auxin; Mesocarp disc; Peach
1. Introduction
To study the growth-promoting effects of auxin, Nitch (1950) nondestructively
removed the achenes of strawberries from the receptacle or emasculated them and
*
Tel.: 81-298-38-6462; fax: 81-298-38-6437.
E-mail address: [email protected] (A. Ohmiya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 3 7 - 5
310
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
left them unpollinated. He applied various kinds of growth regulators to the
deachened receptacles and found that auxin was the only hormone that would
sustain normal fruit growth. Nitch's classic study clearly demonstrated that auxin
plays an important role in fruit enlargement and that seeds are a good source of
auxin in fruit ¯esh. In most fruits, however, it is extremely dif®cult to remove
seeds nondestructively to allow an unambiguous study of responses to
exogenously applied auxin. Therefore, little experimental data are available on
effects of auxin on fruit enlargement. Anatomically, the strawberry is not a true
fruit, since it is derived from receptacle tissue rather than ovary itself. Little
emphasis has been placed on determining auxin effects on mesocarp tissues of
fruits. Recently, several attempts have been made to examine physiological
changes in vitro using fruit discs (Parkin, 1987; Campbell et al., 1990) and whole
fruit (Cohen, 1996; Perkins-Veazie et al., 1996). Parkin (1987) showed that the
ripening phenomena observed in pericarp discs of tomato fruit at green mature
stage were temporally associated with intact tomato fruit over a 30-day period. In
the present study, peach fruit ¯esh, which is derived from mesocarp, was chosen
as the plant material and a model system for the experimental analyses of auxin
effects was established using excised mesocarp discs of peach.
Peach fruit display a double sigmoidal growth curve. This phasic pattern of
growth is customarily divided into three stages: FWI, initial period of exponential
growth; FWII, period of slow growth; and FWIII, second period of exponential
growth. Miller et al. (1987) showed that IAA concentrations in peach fruit are
relatively high at FWI and FWIII. IAA concentrations reach their lowest levels
during the lag phase (FWII) of peach fruit growth. These data imply that IAA
serves as a signi®cant growth promoter during both FWI and FWIII.
In present study, to study the physiological effects of auxin on fruit ¯esh of
peach, mesocarp discs were prepared from peach fruit at FWI, FWII, and FWIII,
and various concentrations of auxin were applied to the discs.
2. Materials and methods
2.1. Growth curve and IAA measurement
Peach fruit (Prunus persica L. cv. Akatsuki) were harvested from trees growing
at the experimental orchard of National Institute of Fruit Tree Science, Tsukuba,
Japan. The growth rate (a) of peach fruit was calculated from the following
equation:
a
log w2 ÿ log w1
w1 w2 t2 ÿ t1
where t2 and t1 are the days after anthesis, and w2 and w1 are the weights of the
fruit at t2 and t1, respectively.
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
311
IAA concentrations of peach fruit were measured according to the method
of Ohmiya and Hayashi (1992). Fruit tissue and indole-3-propionic acid (internal
standard) were added to 65% isopropanol±imidazole buffer (pH 7.0), followed by
homogenization. Acidic compounds were obtained by three extractions with
dichloromethane at pH 2.0. The dichloromethane fraction was extracted with
imidazole buffer (pH 7.0). The aqueous phase was applied to a `Baker'-10SPE
NH2 column (J.T. Baker, New Jersey, USA) and eluted with 5% acetic acid
in methanol. The fractions containing IAA were dried in vacuum, redissolved
in 7% methanol and subjected to HPLC (Shimazu LC-9A, Kyoto, Japan)
with a reverse phase C-18 column and a ¯uorometric detector. At each sampling
point a minimum of 10 fruit were subjected to measurement of auxin concentration.
2.2. Preparation of mesocarp discs and application of auxin
Peach fruit were harvested at three stages of development: 38 days (FWI), 60
days (FWII), and 88 days (FWIII) after anthesis. Fruit was peeled and surfacesterilized in a solution of 7% NaOCl containing 50 mg/l of Tween 20 for 12 min,
followed by three rinses with sterile water. Tissue cylinders (10 mm in diameter)
were excised from the mesocarp with a cork borer, and 3 mm thick discs were cut
with a razor blade from the cylinders. They were incubated with the skin side up
under sterile conditions in 90 mm 20 mm petri dishes containing 25 ml of MS
medium (Murashige and Skoog, 1962), pH 5.8, supplemented with various
concentrations of 1-naphthaleneacetic acid (NAA) and 3% (w/v) sucrose, and
solidi®ed with 0.9% (w/v) agar. More than 100 discs were prepared for each
concentration of NAA. Incubations were performed in a growth cabinet at 258C
under a 16/8 h light/dark cycle (5000 lx) or under conditions of constant darkness.
Mechanical wounding has been shown to cause fruit tissue to produce large
amount of ethylene during several hours (or days) following excision (Yu and
Yang, 1980). Such enhanced rates of respiration upon cutting slices were also
reported for tomato fruit (Parkin, 1987; Campbell et al., 1990). Campbell et al.
(1990) found that the wound ethylene induced by cutting of tomato pericarp discs
abated within 48 h. Therefore, petri dishes were left uncovered for 10 min/day
during the 7-day incubation period to ¯ush the air, thus reducing the effects of
wound-induced changes in atmospheric conditions.
2.3. Measurement of ®rmness
Firmness of the discs was measured using a Reona-RE-3305 (Yamaden Co.
Ltd., Tokyo, Japan) ®tted with a 3 mm probe and 2 or 20 kg of load cell. The
probe was driven 2.0 mm into the discs, and the peak compression force was
recorded. Firmness of twenty discs were measured at each NAA concentration
312
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
after 1 week and 4 weeks of incubation. P-values were calculated based on
student's t-test.
2.4. Measurement of anthocyanin concentration
Mesocarp discs (1±3 g) were ground in methanol containing 1% (v/v) HCl, and
were centrifuged at 10 000 g. Anthocyanin content was determined by measuring
the absorbance at 528 nm of the supernatant and was expressed as mg of
keracyanin per gfw of the discs. Keracyanin used for the standard curve has a
structure similar to that of cyanidin-3-monoglucoside, which is contained in
peach fruit (Blaricom and Senn, 1967). Four discs were subjected to anthocyanin
measurement after 4 weeks of incubation.
3. Results
3.1. Growth curve of Akatsuki fruit and IAA content
There was a sigmoidal relationship between fruit weight and the time of
development in Akatsuki cultivar (Fig. 1A). Growth of Akatsuki fruit was divided
as follows into three stages according to the growth curve and the time of
hardening of the endocarp; FWI, 0±50 days; FWII, 51±83 days; and FWIII, 84±
105 days after anthesis. Peaks in the growth rate occurred at FWI (Fig. 1B).
Growth rate was lowest at FWII, and increased slightly at FWIII.
IAA concentration ¯uctuated drastically during peach fruit development; peaks
occurred at 5 and 105 (time of harvest) days after anthesis with 12.5 and 21.1 ng/
gfw, respectively (Fig. 1B). The IAA concentration value was extremely low
during FWII. The pattern well correlated with the growth rate of Akatsuki fruit,
except the drastic increase of IAA concentration just prior to maturity.
3.2. Incubation of mesocarp discs with NAA
Mesocarp discs excised aseptically from peach fruit can be used for long-term
incubation (4 weeks) on medium, both with and without auxin. The system used
here was found to be useful for examining the effects of auxin on physiological
and morphological changes in discs, since there was little variation among discs
in response to auxin. Since the results obtained were almost identical, regardless
of whether NAA or 2,4-D was used as an auxin, only data using NAA were
presented in this paper. The discs incubated on medium containing more than
500 mM of NAA showed necrosis. There were few differences between discs
incubated with less than 0.001 mM of NAA and discs incubated without NAA.
Therefore, NAA concentrations from 0.01 to 100 mM were used for the present
experiment.
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
313
Fig. 1. (A): Fresh weight growth curve of peach fruit cv. Akatsuki. (B): IAA concentration ((*)
whole fruit; (*) mesocarp) and growth rate (open bars) of developing peach fruit cv. Akatsuki.
At 105 days of anthesis, fruit was fully expanded and capable of softening to a fully ripe stage
within a few days of harvest. Fruit harvested at 105 days after anthesis were stored at 258C for
3 days (fully ripe), and then at 58C for 4 days (expressed as fruit at 112 days after anthesis in
Fig. 1(A) and 1(B).
FWI discs at the time of sampling and incubated in light for 4 weeks with
various concentrations of NAA are presented in Fig. 2. The discs incubated with
1±100 mM of NAA were signi®cantly larger than discs incubated without NAA.
In addition, discs incubated with 1±100 mM of NAA formed more anthocyanin at
the surface than did discs incubated without NAA. Enlargement of discs was
almost completed after 3 weeks when ripening processes such as softening and
anthocyanin formation were still in progress.
3.3. The effect of NAA on the enlargement of discs
Fig. 3 shows the effect of NAA on the weight of mesocarp discs of FWI, FWII,
and FWIII incubated under light. The weight of FWI, FWII, and FWIII discs
incubated without NAA increased gradually, reaching 2.4-, 2.7-, and 3.5-fold,
314
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
Fig. 2. Photographs of FWI peach mesocarp discs (A): at the time of sampling; (B): incubated in
the light for 4 weeks with various concentrations of NAA. 1, control (without NAA); 2, 0.01 mM
NAA; 3, 0.1 mM NAA; 4, 1 mM NAA; 5, 10 mM NAA; and 6, 100 mM NAA.
respectively, after 4 weeks. When the discs were incubated on medium containing
NAA, the weights of FWI and FWII discs were signi®cantly higher than those of
discs incubated without NAA. The highest value of the weight of FWI discs was
Fig. 3. Effects of NAA on the weight of peach mesocarp discs (A): incubated for 1 week in light;
(B): incubated 4 weeks in light; (*) FWI; (&) FWII; and (~) FWIII. Standard errors (vertical
bars) within 0.01 g were omitted from the ®gure (n 20).
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
315
observed at 10 mM of NAA (1.52 0.039 g) and that of FWII discs was at 1 mM
(1.48 0.015 g). Weights increased 3.1-fold at FWI (P < 0.001) and 2.7-fold at
FWII (P < 0.001), respectively, compared to discs incubated without NAA.
Although FWIII discs incubated with NAA for 1 week were signi®cantly larger
than those without NAA, they stopped growing thereafter and marked difference
was not observed among NAA concentrations after 4 weeks. The effects of NAA
on the weight of discs incubated in darkness showed a similar pattern to that of
discs incubated in light, showing maximum values of FWI discs at 1 mM of NAA
(1.01 0.025 g) and FWII discs at 10 mM (1.21 0.012 g). These values were
signi®cantly lower than those in light (P < 0.01).
3.4. The effect of NAA on the ®rmness of discs
Firmness of discs at the time they were taken was 218.1 8.7 g (FWI),
212.3 11.5 g (FWII), and 135.9 5.1 g (FWIII). After 4 weeks of incubation,
there was a slight decrease in the ®rmness of FWI and FWII discs on medium
without NAA, showing 126.3 15.7 g and 83.8 17.4 g, respectively (Fig. 4).
Lower values were observed with increasing concentrations of NAA up to
100 mM in both FWI and FWII discs; the ®rmness of FWII discs was lower than
that of FWI discs at each concentration of NAA (P < 0.01). The ®rmness of
FWIII discs was signi®cantly lower than that of both FWI and FWII discs at the
time of sampling (P < 0.01). After 1 week of incubation, the FWIII discs treated
with NAA showed lower values than those of discs incubated without NAA
(P < 0.01). After 4 weeks, the discs showed extremely low levels of ®rmness,
both with and without NAA, and no signi®cant difference existed among them.
There was no signi®cant difference in ®rmness between light- and dark-incubated
discs (data not shown).
Fig. 4. Effects of NAA on ®rmness of peach mesocarp discs (A): incubated for 1 week in light; (B):
incubated for 4 weeks in light; (*) FWI; (&) FWII; and (~) FWIII). Standard errors (vertical
bars) within 0.01 g were omitted from the ®gure (n 20).
316
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
Fig. 5. Effects of NAA on the anthocyanin content of discs (A): incubated for 1 week in light; (B):
incubated for 1 week in darkness; (C): incubated for 4 weeks in light; (D): incubated for 4 weeks in
darkness; (*) FWI; (&) FWII; and (~) FWIII). Standard errors (vertical bars) within 0.1 mg/gFW
were omitted from the ®gure (n 4).
3.5. The effect of NAA on anthocyanin formation
There was a remarkable difference in anthocyanin formation between light- and
dark-incubated discs. When FWI and FWII discs were incubated under light,
anthocyanin formation was limited to the surface of discs, showing their highest
value at 10 mM of NAA (Fig. 5). There was a higher level of anthocyanin
formation in FWII discs as compared to FWI discs at 1±100 mM of NAA
(P < 0.05). In dark conditions, anthocyanin formation occurred inside the discs
without NAA or in those with low concentrations (0.01±0.1 mM) of NAA. On the
other hand, high concentrations (10±100 mM) of NAA stimulated anthocyanin
formation at the surface of the discs. Whereas very little anthocyanin was formed
in both FWI and FWII discs within 1 week, relatively high levels of anthocyanin
were formed in the FWIII discs; the maximum level was observed at 0.1 mM of
NAA. After 4 weeks, high levels of anthocyanin were formed regardless of NAA
concentrations and conditions of light.
Anthocyanin developed in mesocarp discs was spectroscopically the same as
that in whole fruit (data not shown). However, the anthocyanin level in the discs
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
317
was much higher in discs than it was in intact fruit. Whole discs were uniformly
stained with anthocyanin, whereas only a small portion of fruit ¯esh was stained
with anthocyanin.
4. Discussion
When treating the whole fruit with auxin, it is dif®cult to equate responses to
externally applied auxins with those by endogenous auxins. Other endogenous
regulators can also interact with this process, rendering it even more dif®cult to
evaluate responses. In addition, it is dif®cult to apply auxin uniformly to whole
fruit. In the present study, excised mesocarp tissue was used to solve these
problems in order to determine the effects of auxin on fruit tissue. The bene®ts of
the present method are as follows: (1) one can separate the effects of regulators
and developmental processes by isolation of speci®c tissues; (2) the quantitative
addition of auxin to the tissues is made possible; (3) conditions such as light,
temperature and nutrition can be uniformly controlled; and (4) variation among
samples can be reduced by taking several samples from the same fruit. In
particular, this system can reduce the effects of endogenous auxin by removing
mesocarp tissue from the presumed source of endogenous auxin. After one or two
weeks, signi®cant changes in physiology and morphology appear in tissue slices
incubated on medium containing NAA.
In the present study, developing fruit was used to examine the effects of auxin
on the growth of discs. Auxin stimulated the enlargement of discs obtained at
FWI and FWII; discs reached approximately three times the size of discs
incubated without auxin. These results suggest that the mesocarp tissue at both
FWI and FWII has the ability to enlarge rapidly when an excess amount of auxin
is supplied. Discs at FWI and FWII were obtained at 36 and 57 days after
anthesis, respectively, when growth rates and endogenous IAA concentrations
were extremely low. This suggests that, at these stages, law auxin concentration is
one of the factors limiting the mesocarp enlargement of attached fruits.
In peach fruit discs, higher concentrations of NAA were needed to bring highest
disc weight at FWI than at FWII. This result suggests that an alteration in the rate
of uptake and/or an alteration in sensitivity to auxin may occur during fruit
development. Changes in sensitivity to auxin during cell differentiation have also
been reported in wheat leaf cells (Wernicke et al., 1986; Wernicke and Milkovits,
1987). Higher concentrations were needed to stimulate cell division in vitro from
more mature regions higher up in the leaf. In addition, such studies have shown
that neither alterations in uptake rate nor alterations in metabolism could account
for the loss of responsiveness to auxin.
Ripening processes such as anthocyanin formation and softening occurred
much earlier in discs than in intact fruit both with and without NAA. On the other
hand, Parkin (1987) used pericarp discs of tomato fruit at green mature stage, and
318
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
showed that the changes in color, ®rmness, and respiratory activity of discs were
temporally associated with intact tomato fruit over a 30-day period. Differences
in responses of these discs are partly due to differences in the developmental
stages of fruit used in these experiments. Parkin (1987) used fully expanded
tomato fruit, while in the present study, enlarging fruit was analyzed. Two
possible mechanisms for triggering the ripening process in fruit discs are
postulated. First, the ripening process of fruit ¯esh may be inhibited by some
factor, which is probably supplied by other parts of the fruit until the cell size of
the fruit reaches its maximum level. Once the tissue is excised from the whole
fruit, this factor is decreased to some extent, and the ripening process may
progress more rapidly. Second, signals may trigger the ripening processes; such
signals would be produced when the cell size reaches its maximum level. It is
assumed that the enlargement of cells in intact peach fruit is partly limited by the
skin, and may progress slower than in discs, resulting in a slower initiation of
ripening in intact fruit. A shorter period was needed for triggering ripening in
discs obtained at a later stage of development. This phenomenon implies that fruit
cells at a later stage of development enter into the ripening stage, i.e., cell size
reaches its maximum limit and/or the ripening inhibiting factor decreases, faster
than it does at an earlier stage.
There was a marked decrease in the ®rmness of discs with increasing amounts
of NAA. Downs et al. (1992) reported that the ®rmness of peach mesocarp was
controlled by polygalacturonase, which is induced by ethylene during ripening
(Grierson and Tucker, 1983). The rate of ethylene production is thought to be
regulated by internal levels of free auxin (Yang and Hoffman, 1984). Accordingly,
higher rates of ethylene production are often associated with those tissues which
contain higher amounts of auxin in vegetative tissues. In fruit tissue, however,
only a few data are available concerning the induction of ethylene by auxin
(Nakagawa et al., 1991). It is therefore necessary to measure the rate of ethylene
formation in discs of peach fruit in order to demonstrate whether or not loss of
®rmness is a direct effect of auxin or if, on the other hand, they are caused
indirectly by ethylene.
Anthocyanin formation is affected by a number of factors including light,
temperature, growth regulators, and developmental stage (Jones, 1984). In peach
mesocarp discs, there was a signi®cant difference in anthocyanin formation
among these conditions; the amount of auxin, with and without light and
developmental stage of fruit, all affected anthocyanin formation. It is worthwhile
to note that the surfaces and insides of discs showed different patterns of
anthocyanin formation in response to light and auxin. Anthocyanin formation at
the surface was stimulated, whereas that inside of discs was inhibited by these
factors. These results suggest that at least two different mechanisms of regulation
of anthocyanin formation may exist in peach mesocarp tissue. Furthermore, such
activity seems to be regulated independently by several factors.
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
319
In conclusion, the present study shows that the bio-assay system using
mesocarp discs of peach is useful for examining long-term effects of auxin on
fruit tissue. Information on the rates of uptake and metabolism of auxin by discs
will provide us with further insight into the relationship between the amount of
auxin and physiological responses of the tissue.
References
Blaricom, L.O.V., Senn, T.L., 1967. Anthocyanin pigment in freestone peaches grown in the
southeast. Amer. Soc. Hort. Sci. 90, 541±545.
Campbell, A.D., Huysamer, M., Stotz, H.U., Greve, L.C., Labavitch, J.M., 1990. Comparison of
ripening processes in intact tomato fruit and excised pericarp discs. Plant Physiol. 94, 1582±
1589.
Cohen, J.D., 1996. In vitro tomato fruit cultures demonstrate a role for indole-3-acetic acid in
regulating fruit ripening. J. Amer. Soc. Hort. Sci. 121, 520±524.
Downs, C.G., Brady, C.G., Gooley, A., 1992. Exopolygalacturonase protein accumulates late in
peach fruits ripening. Physiol. Plant. 85, 133±140.
Grierson, D., Tucker, G.A., 1983. Timing of ethylene and polygalacturonase synthesis in relation to
the control of tomato fruit ripening. Planta 157, 174±179.
Jones, D.H., 1984. Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant
development. Phytochemistry 23, 1349±1359.
Miller, A.N., Walsh, C.S., Cohen, J.D., 1987. Measurement of indole-3-acetic acid in peach fruits
(Prunus persica L. Batch cv.Redhaven) during development. Plant Physiol. 84, 491±494.
Murashige, T.M., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco
tissue cultures. Physiol. Plant. 15, 473±497.
Nakagawa, N., Mori, H., Yamazaki, K., Imaseki, H., 1991. Cloning of a complementary DNA for
auxin-induced 1-aminocyclopropane-1-carboxylate synthase and differential expression of the
gene by auxin and wounding. Plant Cell Physiol. 32, 1153±1163.
Nitch, J.P., 1950. Growth and morphogenesis of the strawberry as related to auxin. Amer. J. Bot. 37,
211±215.
Ohmiya, A., Hayashi, T., 1992. Immuno-gold localization of IAA in leaf cells of Prunus persica at
different stages of development. Physiol. Plant. 85, 439±445.
Parkin, K.L., 1987. A new technique for the long-term study of physiology of plant fruit tissue
slices. Physiol. Plant. 69, 472±476.
Perkins-Veazie, P.M., Huber, D.J., Brecht, J.K., 1996. In vitro growth and ripening of strawberry
fruit in the presence of ACC, STS or propylene. Ann. Appl. Biol. 128, 105±116.
Wernicke, W., Gorst, J., Milkovits, L., 1986. The ambiguous role of 2,4-dichlorophenoxyacetic acid
in wheat tissue culture. Physiol. Plant. 68, 597±602.
Wernicke, W., Milkovits, L., 1987. Rates of uptake and metabolism of indole-3-acetic acid and 2,4dichlorophenoxyacetic acid by cultured leaf segments at different stages of development in
wheat. Physiol. Plant. 69, 23±28.
Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu.
Rev. Plant Physiol. 35, 155±189.
Yu, Y-B., Yang, S.F., 1980. Biosynthesis of wound ethylene. Plant Physiol. 66, 281±285.
Effects of auxin on growth and ripening
of mesocarp discs of peach fruit
Akemi Ohmiya*
National Institute of Fruit Tree Science, Ministry of Agriculture,
Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan
Accepted 8 November 1999
Abstract
A bio-assay system using the mesocarp discs of peach fruit (Prunus persica L. cv. Akatsuki) was
developed, and the effects of auxin on the physiology of fruit tissues were investigated at different
stages of development (FWI: initial period of exponential growth, FWII: period of slow growth, and
FWIII: second period of exponential growth). Auxin promoted both the enlargement of discs as well
as ripening processes such as softening and anthocyanin formation. In particular, discs at FWI and
FWII enlarged remarkably after four weeks of incubation with NAA. The highest value of the
weight of FWI discs was observed at 10 mM of NAA; that of FWII discs was at 1 mM. Weights were
compared between discs that were incubated with and without NAA. NAA-incubated discs reached
to 3.1-fold in the FWI stage and 2.7-fold in the FWII stage. Discs at each stage of development lost
their ®rmness with increasing concentrations of NAA up to 100 mM. There was a signi®cant
difference in anthocyanin formation between light- and dark-incubated discs. Anthocyanin
formation at the surface of the discs was enhanced by high concentrations of NAA and light,
whereas that inside of the discs was enhanced by darkness and low concentrations of NAA. These
multiple effects of auxin on growth and the ripening process of fruit tissue may be caused by
multiple mechanisms of auxin action as in¯uenced by the stage of fruit tissue growth and
environmental conditions. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Auxin; Mesocarp disc; Peach
1. Introduction
To study the growth-promoting effects of auxin, Nitch (1950) nondestructively
removed the achenes of strawberries from the receptacle or emasculated them and
*
Tel.: 81-298-38-6462; fax: 81-298-38-6437.
E-mail address: [email protected] (A. Ohmiya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 3 7 - 5
310
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
left them unpollinated. He applied various kinds of growth regulators to the
deachened receptacles and found that auxin was the only hormone that would
sustain normal fruit growth. Nitch's classic study clearly demonstrated that auxin
plays an important role in fruit enlargement and that seeds are a good source of
auxin in fruit ¯esh. In most fruits, however, it is extremely dif®cult to remove
seeds nondestructively to allow an unambiguous study of responses to
exogenously applied auxin. Therefore, little experimental data are available on
effects of auxin on fruit enlargement. Anatomically, the strawberry is not a true
fruit, since it is derived from receptacle tissue rather than ovary itself. Little
emphasis has been placed on determining auxin effects on mesocarp tissues of
fruits. Recently, several attempts have been made to examine physiological
changes in vitro using fruit discs (Parkin, 1987; Campbell et al., 1990) and whole
fruit (Cohen, 1996; Perkins-Veazie et al., 1996). Parkin (1987) showed that the
ripening phenomena observed in pericarp discs of tomato fruit at green mature
stage were temporally associated with intact tomato fruit over a 30-day period. In
the present study, peach fruit ¯esh, which is derived from mesocarp, was chosen
as the plant material and a model system for the experimental analyses of auxin
effects was established using excised mesocarp discs of peach.
Peach fruit display a double sigmoidal growth curve. This phasic pattern of
growth is customarily divided into three stages: FWI, initial period of exponential
growth; FWII, period of slow growth; and FWIII, second period of exponential
growth. Miller et al. (1987) showed that IAA concentrations in peach fruit are
relatively high at FWI and FWIII. IAA concentrations reach their lowest levels
during the lag phase (FWII) of peach fruit growth. These data imply that IAA
serves as a signi®cant growth promoter during both FWI and FWIII.
In present study, to study the physiological effects of auxin on fruit ¯esh of
peach, mesocarp discs were prepared from peach fruit at FWI, FWII, and FWIII,
and various concentrations of auxin were applied to the discs.
2. Materials and methods
2.1. Growth curve and IAA measurement
Peach fruit (Prunus persica L. cv. Akatsuki) were harvested from trees growing
at the experimental orchard of National Institute of Fruit Tree Science, Tsukuba,
Japan. The growth rate (a) of peach fruit was calculated from the following
equation:
a
log w2 ÿ log w1
w1 w2 t2 ÿ t1
where t2 and t1 are the days after anthesis, and w2 and w1 are the weights of the
fruit at t2 and t1, respectively.
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311
IAA concentrations of peach fruit were measured according to the method
of Ohmiya and Hayashi (1992). Fruit tissue and indole-3-propionic acid (internal
standard) were added to 65% isopropanol±imidazole buffer (pH 7.0), followed by
homogenization. Acidic compounds were obtained by three extractions with
dichloromethane at pH 2.0. The dichloromethane fraction was extracted with
imidazole buffer (pH 7.0). The aqueous phase was applied to a `Baker'-10SPE
NH2 column (J.T. Baker, New Jersey, USA) and eluted with 5% acetic acid
in methanol. The fractions containing IAA were dried in vacuum, redissolved
in 7% methanol and subjected to HPLC (Shimazu LC-9A, Kyoto, Japan)
with a reverse phase C-18 column and a ¯uorometric detector. At each sampling
point a minimum of 10 fruit were subjected to measurement of auxin concentration.
2.2. Preparation of mesocarp discs and application of auxin
Peach fruit were harvested at three stages of development: 38 days (FWI), 60
days (FWII), and 88 days (FWIII) after anthesis. Fruit was peeled and surfacesterilized in a solution of 7% NaOCl containing 50 mg/l of Tween 20 for 12 min,
followed by three rinses with sterile water. Tissue cylinders (10 mm in diameter)
were excised from the mesocarp with a cork borer, and 3 mm thick discs were cut
with a razor blade from the cylinders. They were incubated with the skin side up
under sterile conditions in 90 mm 20 mm petri dishes containing 25 ml of MS
medium (Murashige and Skoog, 1962), pH 5.8, supplemented with various
concentrations of 1-naphthaleneacetic acid (NAA) and 3% (w/v) sucrose, and
solidi®ed with 0.9% (w/v) agar. More than 100 discs were prepared for each
concentration of NAA. Incubations were performed in a growth cabinet at 258C
under a 16/8 h light/dark cycle (5000 lx) or under conditions of constant darkness.
Mechanical wounding has been shown to cause fruit tissue to produce large
amount of ethylene during several hours (or days) following excision (Yu and
Yang, 1980). Such enhanced rates of respiration upon cutting slices were also
reported for tomato fruit (Parkin, 1987; Campbell et al., 1990). Campbell et al.
(1990) found that the wound ethylene induced by cutting of tomato pericarp discs
abated within 48 h. Therefore, petri dishes were left uncovered for 10 min/day
during the 7-day incubation period to ¯ush the air, thus reducing the effects of
wound-induced changes in atmospheric conditions.
2.3. Measurement of ®rmness
Firmness of the discs was measured using a Reona-RE-3305 (Yamaden Co.
Ltd., Tokyo, Japan) ®tted with a 3 mm probe and 2 or 20 kg of load cell. The
probe was driven 2.0 mm into the discs, and the peak compression force was
recorded. Firmness of twenty discs were measured at each NAA concentration
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after 1 week and 4 weeks of incubation. P-values were calculated based on
student's t-test.
2.4. Measurement of anthocyanin concentration
Mesocarp discs (1±3 g) were ground in methanol containing 1% (v/v) HCl, and
were centrifuged at 10 000 g. Anthocyanin content was determined by measuring
the absorbance at 528 nm of the supernatant and was expressed as mg of
keracyanin per gfw of the discs. Keracyanin used for the standard curve has a
structure similar to that of cyanidin-3-monoglucoside, which is contained in
peach fruit (Blaricom and Senn, 1967). Four discs were subjected to anthocyanin
measurement after 4 weeks of incubation.
3. Results
3.1. Growth curve of Akatsuki fruit and IAA content
There was a sigmoidal relationship between fruit weight and the time of
development in Akatsuki cultivar (Fig. 1A). Growth of Akatsuki fruit was divided
as follows into three stages according to the growth curve and the time of
hardening of the endocarp; FWI, 0±50 days; FWII, 51±83 days; and FWIII, 84±
105 days after anthesis. Peaks in the growth rate occurred at FWI (Fig. 1B).
Growth rate was lowest at FWII, and increased slightly at FWIII.
IAA concentration ¯uctuated drastically during peach fruit development; peaks
occurred at 5 and 105 (time of harvest) days after anthesis with 12.5 and 21.1 ng/
gfw, respectively (Fig. 1B). The IAA concentration value was extremely low
during FWII. The pattern well correlated with the growth rate of Akatsuki fruit,
except the drastic increase of IAA concentration just prior to maturity.
3.2. Incubation of mesocarp discs with NAA
Mesocarp discs excised aseptically from peach fruit can be used for long-term
incubation (4 weeks) on medium, both with and without auxin. The system used
here was found to be useful for examining the effects of auxin on physiological
and morphological changes in discs, since there was little variation among discs
in response to auxin. Since the results obtained were almost identical, regardless
of whether NAA or 2,4-D was used as an auxin, only data using NAA were
presented in this paper. The discs incubated on medium containing more than
500 mM of NAA showed necrosis. There were few differences between discs
incubated with less than 0.001 mM of NAA and discs incubated without NAA.
Therefore, NAA concentrations from 0.01 to 100 mM were used for the present
experiment.
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
313
Fig. 1. (A): Fresh weight growth curve of peach fruit cv. Akatsuki. (B): IAA concentration ((*)
whole fruit; (*) mesocarp) and growth rate (open bars) of developing peach fruit cv. Akatsuki.
At 105 days of anthesis, fruit was fully expanded and capable of softening to a fully ripe stage
within a few days of harvest. Fruit harvested at 105 days after anthesis were stored at 258C for
3 days (fully ripe), and then at 58C for 4 days (expressed as fruit at 112 days after anthesis in
Fig. 1(A) and 1(B).
FWI discs at the time of sampling and incubated in light for 4 weeks with
various concentrations of NAA are presented in Fig. 2. The discs incubated with
1±100 mM of NAA were signi®cantly larger than discs incubated without NAA.
In addition, discs incubated with 1±100 mM of NAA formed more anthocyanin at
the surface than did discs incubated without NAA. Enlargement of discs was
almost completed after 3 weeks when ripening processes such as softening and
anthocyanin formation were still in progress.
3.3. The effect of NAA on the enlargement of discs
Fig. 3 shows the effect of NAA on the weight of mesocarp discs of FWI, FWII,
and FWIII incubated under light. The weight of FWI, FWII, and FWIII discs
incubated without NAA increased gradually, reaching 2.4-, 2.7-, and 3.5-fold,
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Fig. 2. Photographs of FWI peach mesocarp discs (A): at the time of sampling; (B): incubated in
the light for 4 weeks with various concentrations of NAA. 1, control (without NAA); 2, 0.01 mM
NAA; 3, 0.1 mM NAA; 4, 1 mM NAA; 5, 10 mM NAA; and 6, 100 mM NAA.
respectively, after 4 weeks. When the discs were incubated on medium containing
NAA, the weights of FWI and FWII discs were signi®cantly higher than those of
discs incubated without NAA. The highest value of the weight of FWI discs was
Fig. 3. Effects of NAA on the weight of peach mesocarp discs (A): incubated for 1 week in light;
(B): incubated 4 weeks in light; (*) FWI; (&) FWII; and (~) FWIII. Standard errors (vertical
bars) within 0.01 g were omitted from the ®gure (n 20).
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
315
observed at 10 mM of NAA (1.52 0.039 g) and that of FWII discs was at 1 mM
(1.48 0.015 g). Weights increased 3.1-fold at FWI (P < 0.001) and 2.7-fold at
FWII (P < 0.001), respectively, compared to discs incubated without NAA.
Although FWIII discs incubated with NAA for 1 week were signi®cantly larger
than those without NAA, they stopped growing thereafter and marked difference
was not observed among NAA concentrations after 4 weeks. The effects of NAA
on the weight of discs incubated in darkness showed a similar pattern to that of
discs incubated in light, showing maximum values of FWI discs at 1 mM of NAA
(1.01 0.025 g) and FWII discs at 10 mM (1.21 0.012 g). These values were
signi®cantly lower than those in light (P < 0.01).
3.4. The effect of NAA on the ®rmness of discs
Firmness of discs at the time they were taken was 218.1 8.7 g (FWI),
212.3 11.5 g (FWII), and 135.9 5.1 g (FWIII). After 4 weeks of incubation,
there was a slight decrease in the ®rmness of FWI and FWII discs on medium
without NAA, showing 126.3 15.7 g and 83.8 17.4 g, respectively (Fig. 4).
Lower values were observed with increasing concentrations of NAA up to
100 mM in both FWI and FWII discs; the ®rmness of FWII discs was lower than
that of FWI discs at each concentration of NAA (P < 0.01). The ®rmness of
FWIII discs was signi®cantly lower than that of both FWI and FWII discs at the
time of sampling (P < 0.01). After 1 week of incubation, the FWIII discs treated
with NAA showed lower values than those of discs incubated without NAA
(P < 0.01). After 4 weeks, the discs showed extremely low levels of ®rmness,
both with and without NAA, and no signi®cant difference existed among them.
There was no signi®cant difference in ®rmness between light- and dark-incubated
discs (data not shown).
Fig. 4. Effects of NAA on ®rmness of peach mesocarp discs (A): incubated for 1 week in light; (B):
incubated for 4 weeks in light; (*) FWI; (&) FWII; and (~) FWIII). Standard errors (vertical
bars) within 0.01 g were omitted from the ®gure (n 20).
316
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
Fig. 5. Effects of NAA on the anthocyanin content of discs (A): incubated for 1 week in light; (B):
incubated for 1 week in darkness; (C): incubated for 4 weeks in light; (D): incubated for 4 weeks in
darkness; (*) FWI; (&) FWII; and (~) FWIII). Standard errors (vertical bars) within 0.1 mg/gFW
were omitted from the ®gure (n 4).
3.5. The effect of NAA on anthocyanin formation
There was a remarkable difference in anthocyanin formation between light- and
dark-incubated discs. When FWI and FWII discs were incubated under light,
anthocyanin formation was limited to the surface of discs, showing their highest
value at 10 mM of NAA (Fig. 5). There was a higher level of anthocyanin
formation in FWII discs as compared to FWI discs at 1±100 mM of NAA
(P < 0.05). In dark conditions, anthocyanin formation occurred inside the discs
without NAA or in those with low concentrations (0.01±0.1 mM) of NAA. On the
other hand, high concentrations (10±100 mM) of NAA stimulated anthocyanin
formation at the surface of the discs. Whereas very little anthocyanin was formed
in both FWI and FWII discs within 1 week, relatively high levels of anthocyanin
were formed in the FWIII discs; the maximum level was observed at 0.1 mM of
NAA. After 4 weeks, high levels of anthocyanin were formed regardless of NAA
concentrations and conditions of light.
Anthocyanin developed in mesocarp discs was spectroscopically the same as
that in whole fruit (data not shown). However, the anthocyanin level in the discs
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
317
was much higher in discs than it was in intact fruit. Whole discs were uniformly
stained with anthocyanin, whereas only a small portion of fruit ¯esh was stained
with anthocyanin.
4. Discussion
When treating the whole fruit with auxin, it is dif®cult to equate responses to
externally applied auxins with those by endogenous auxins. Other endogenous
regulators can also interact with this process, rendering it even more dif®cult to
evaluate responses. In addition, it is dif®cult to apply auxin uniformly to whole
fruit. In the present study, excised mesocarp tissue was used to solve these
problems in order to determine the effects of auxin on fruit tissue. The bene®ts of
the present method are as follows: (1) one can separate the effects of regulators
and developmental processes by isolation of speci®c tissues; (2) the quantitative
addition of auxin to the tissues is made possible; (3) conditions such as light,
temperature and nutrition can be uniformly controlled; and (4) variation among
samples can be reduced by taking several samples from the same fruit. In
particular, this system can reduce the effects of endogenous auxin by removing
mesocarp tissue from the presumed source of endogenous auxin. After one or two
weeks, signi®cant changes in physiology and morphology appear in tissue slices
incubated on medium containing NAA.
In the present study, developing fruit was used to examine the effects of auxin
on the growth of discs. Auxin stimulated the enlargement of discs obtained at
FWI and FWII; discs reached approximately three times the size of discs
incubated without auxin. These results suggest that the mesocarp tissue at both
FWI and FWII has the ability to enlarge rapidly when an excess amount of auxin
is supplied. Discs at FWI and FWII were obtained at 36 and 57 days after
anthesis, respectively, when growth rates and endogenous IAA concentrations
were extremely low. This suggests that, at these stages, law auxin concentration is
one of the factors limiting the mesocarp enlargement of attached fruits.
In peach fruit discs, higher concentrations of NAA were needed to bring highest
disc weight at FWI than at FWII. This result suggests that an alteration in the rate
of uptake and/or an alteration in sensitivity to auxin may occur during fruit
development. Changes in sensitivity to auxin during cell differentiation have also
been reported in wheat leaf cells (Wernicke et al., 1986; Wernicke and Milkovits,
1987). Higher concentrations were needed to stimulate cell division in vitro from
more mature regions higher up in the leaf. In addition, such studies have shown
that neither alterations in uptake rate nor alterations in metabolism could account
for the loss of responsiveness to auxin.
Ripening processes such as anthocyanin formation and softening occurred
much earlier in discs than in intact fruit both with and without NAA. On the other
hand, Parkin (1987) used pericarp discs of tomato fruit at green mature stage, and
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showed that the changes in color, ®rmness, and respiratory activity of discs were
temporally associated with intact tomato fruit over a 30-day period. Differences
in responses of these discs are partly due to differences in the developmental
stages of fruit used in these experiments. Parkin (1987) used fully expanded
tomato fruit, while in the present study, enlarging fruit was analyzed. Two
possible mechanisms for triggering the ripening process in fruit discs are
postulated. First, the ripening process of fruit ¯esh may be inhibited by some
factor, which is probably supplied by other parts of the fruit until the cell size of
the fruit reaches its maximum level. Once the tissue is excised from the whole
fruit, this factor is decreased to some extent, and the ripening process may
progress more rapidly. Second, signals may trigger the ripening processes; such
signals would be produced when the cell size reaches its maximum level. It is
assumed that the enlargement of cells in intact peach fruit is partly limited by the
skin, and may progress slower than in discs, resulting in a slower initiation of
ripening in intact fruit. A shorter period was needed for triggering ripening in
discs obtained at a later stage of development. This phenomenon implies that fruit
cells at a later stage of development enter into the ripening stage, i.e., cell size
reaches its maximum limit and/or the ripening inhibiting factor decreases, faster
than it does at an earlier stage.
There was a marked decrease in the ®rmness of discs with increasing amounts
of NAA. Downs et al. (1992) reported that the ®rmness of peach mesocarp was
controlled by polygalacturonase, which is induced by ethylene during ripening
(Grierson and Tucker, 1983). The rate of ethylene production is thought to be
regulated by internal levels of free auxin (Yang and Hoffman, 1984). Accordingly,
higher rates of ethylene production are often associated with those tissues which
contain higher amounts of auxin in vegetative tissues. In fruit tissue, however,
only a few data are available concerning the induction of ethylene by auxin
(Nakagawa et al., 1991). It is therefore necessary to measure the rate of ethylene
formation in discs of peach fruit in order to demonstrate whether or not loss of
®rmness is a direct effect of auxin or if, on the other hand, they are caused
indirectly by ethylene.
Anthocyanin formation is affected by a number of factors including light,
temperature, growth regulators, and developmental stage (Jones, 1984). In peach
mesocarp discs, there was a signi®cant difference in anthocyanin formation
among these conditions; the amount of auxin, with and without light and
developmental stage of fruit, all affected anthocyanin formation. It is worthwhile
to note that the surfaces and insides of discs showed different patterns of
anthocyanin formation in response to light and auxin. Anthocyanin formation at
the surface was stimulated, whereas that inside of discs was inhibited by these
factors. These results suggest that at least two different mechanisms of regulation
of anthocyanin formation may exist in peach mesocarp tissue. Furthermore, such
activity seems to be regulated independently by several factors.
A. Ohmiya / Scientia Horticulturae 84 (2000) 309±319
319
In conclusion, the present study shows that the bio-assay system using
mesocarp discs of peach is useful for examining long-term effects of auxin on
fruit tissue. Information on the rates of uptake and metabolism of auxin by discs
will provide us with further insight into the relationship between the amount of
auxin and physiological responses of the tissue.
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