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 persicaL. 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 10mM of NAA; that of FWII discs was at 1mM. 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 100mM. 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
*
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E-mail address: [email protected] (A. Ohmiya).
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 persicaL. 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 w2ÿlog w1 w1w2 t2ÿt1
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 con-centration.
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 surface-sterilized 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 mm20 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
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
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±100mM of NAA were signi®cantly larger than discs incubated without NAA. In addition, discs incubated with 1±100mM 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,
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. 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, 0.01mM NAA; 3, 0.1mM NAA; 4, 1mM NAA; 5, 10mM NAA; and 6, 100mM NAA.
observed at 10mM of NAA (1.520.039 g) and that of FWII discs was at 1mM (1.480.015 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 1mM of NAA (1.010.025 g) and FWII discs at 10mM (1.210.012 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 218.18.7 g (FWI), 212.311.5 g (FWII), and 135.95.1 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 126.315.7 g and 83.817.4 g, respectively (Fig. 4). Lower values were observed with increasing concentrations of NAA up to 100mM 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).
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 10mM of NAA (Fig. 5). There was a higher level of anthocyanin formation in FWII discs as compared to FWI discs at 1±100mM of NAA (P< 0.05). In dark conditions, anthocyanin formation occurred inside the discs without NAA or in those with low concentrations (0.01±0.1mM) of NAA. On the other hand, high concentrations (10±100mM) 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 0.1mM 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
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.
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.
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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