Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue1.Jan2000:
Scientia Horticulturae 83 (2000) 33±41
Endogenous polyamines in the pericarp and seed of
the grape berry during development and ripening
Shuji Shiozaki*, Tsuneo Ogata, Shosaku Horiuchi
College of Agriculture, Osaka Prefecture University, Gakuencho 1-1, Sakai Osaka 599-8531, Japan
Accepted 9 April 1999
Abstract
The levels of free, perchloric acid-soluble conjugated and perchloric acid-insoluble bound
polyamines were determined in pericarp and seeds of `Muscat Bailey A' grapes (Vitis labrusca
L. Vitis vinifera L.) during development and ripening. In both, the pericarp and seeds, putrescine
was the predominant polyamine in the three fractions, and the bound polyamine level was the
highest of the fractions. In the pericarp, the levels of free putrescine and spermidine were higher
during early development. In all fractions, all polyamines in the pericarp increased 30 days after full
bloom; the increase was greatest in conjugated polyamines, and least in free polyamines. These
increases coincided with an increase in the levels of free polyamines in the seeds. Polyamine levels
in all fractions were almost constant during ripening. In the seed, the levels of free polyamines
increased when the levels of conjugated polyamines decreased at 30 days after full bloom. The
levels of conjugated and bound polyamines increased 50 days after full bloom, with a decrease in
the free polyamine level. The inverse relation between the change in the levels of free polyamine
and of conjugated and/or bound polyamines was a peculiar feature to the seeds. # 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Grape berry; Grape seed; Polyamines; Putrescine; Spermidine; Spermine
1. Introduction
Polyamines (PA) are found in all organisms and are believed to be involved in
several physiological processes in higher plants, including morphogenesis,
* Corresponding author. Tel.: +81-722-549417; fax: +81-722-549417.
E-mail address: shiozaki@plant.osakafu-u.ac.jp (S. Shiozaki)
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 0 6 4 - 3
34
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
rooting, flowering and senescence (Evans and Malmberg, 1989). Although the
precise physiological role of PA in fruit development has not been established,
evidence for its involvement in the development and ripening of fruit arises from
the changes found in PA levels and metabolism in many fruits. These fruits are
divided into two groups according to the changes in PA levels. In the apple (Biasi
et al., 1988), avocado (Kushad et al., 1988), pear (Toumadje and Richardson,
1988), pepper (Serrano et al., 1995) and strawberry (Ponappa and Miller, 1996),
PA levels are high in the early phases of development and gradually decrease as
development progresses. In contrast, in mandarin (Nathan et al., 1984), orange
(Hasdai et al., 1986) and cherimoya (Escribano and Merodio, 1994), an increase
in PA levels is observed during ripening. The changes in PA levels may reflect
features of fruit growth and development in each fruit species.
In fruit, seeds are generally a metabolic center of phytohormones (Nitsch,
1970), so that the influence of seeds must be taken into account in discussions of
fruit development and ripening. The sizes of grapes and kiwifruits are closely
correlated with the numbers of seeds (Lavee, 1960; Pyke and Alspach, 1986). In
addition, Scienza et al. (1978) reported that the greater the number of seeds, the
higher the levels of gibberellin and abscisic acid in the pericarp of grape berries.
Since the phytohormones produced in seeds play a role in development of the
pericarp, PA produced in seeds may act in a similar manner.
Little evidence is currently available regarding the relationships between PA
levels in pericarp and seeds and the development of grape berry. In this study, we
analysed the levels of free, conjugated and bound PA in the pericarp and seeds of
grape berries during development and ripening. Relations between PA levels in
the pericarp and seeds and grape berry development and ripening are discussed.
2. Materials and methods
2.1. Plants
Three vines of four-year-old cv. `Muscat Bailey A' grapes planted in Osaka
Prefecture University were used. To study development of the berry, the width of
10 berries, selected randomly from five clusters and marked with thread, was
measured at 3±9-day intervals, from two days after full bloom (DAB) to harvest.
The fresh weight of 10 seeds taken from berries, which were randomly sampled
from five clusters, was determined at 10±20-day intervals from 20 DAB to
harvest.
Flowers and berries were randomly sampled from five clusters at 0, 10, 20, 30,
40, 50, 70, 90 DAB. On, and after, Day 20, seeds were taken from berries.
Replicate samples (500±700 mg) of flower, pericarp (including seeds in the 10
DAB sample) and seeds were frozen in liquid nitrogen, and stored at ÿ308C.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
35
2.2. Polyamine extraction
The extraction procedure of PA was essentially that of Smith (1991). Samples
were homogenized in cold 10% perchloric acid (PCA) (0.1 g tissue/ml PCA)
using a glass homogenizer, and the homogenate was maintained at 48C for
30 min. The extracts were centrifuged for 20 min at 12 500 g, and the supernatant fraction was used for the determination of free PA and PCA-soluble
conjugated PA. The pellet was used for the determination of PCA-insoluble
bound PA. It was washed in 5 ml of PCA, centrifuged for 20 min at 12 500 g,
then resuspended in the original volume of PCA by vortexing. The pellet
suspension and the original supernatant (0.2 ml each) were hydrolyzed for 18 h
with 0.2 ml of 12N HCl at 1108C in a reaction vial. The hydrolysate was
centrifuged and 0.1 ml aliquot of the supernatant was dried in vacuo at 608C, then
dissolved in 0.1 ml PCA. The soluble conjugated PA was estimated as the
concentration of PA in the hydrolysate of the original supernatant less that of the
free PA.
2.3. Dansylation of polyamines and HPLC analysis
The extracts were dansylated as described by Smith (1991). An aliquot (0.1 ml)
of the extract was added to 0.2 ml saturated sodium carbonate and 0.4 ml dansyl
chloride in acetone (7.5 mg/ml). The mixture was incubated at 608C for 30 min in
the dark. In order to eliminate excess dansyl chloride, 0.1 ml of proline (0.1 g/ml)
was added to the mixture which was incubated at room temperature for 15 min in
the dark. Dansylated PA were extracted with 0.5 ml toluene by vortexing for
1 min, and a 0.2 ml aliquot of toluene was dried. The derivatives were redissolved
in methanol and analyzed by reverse-phase HPLC with a fluorescence detector.
The excision and emission wavelengths were, respectively, 365 and 510 nm.
Samples were eluted from the reversed-phase HPLC column (4.6 mm 250 mm)
using a linear solvent gradient, from 60% methanol in pH 3.5 acetate buffer to
95% methanol, over 25 min, the latter for 10 min at a flow rate of 1 ml/min. Each
determination was performed in triplicate.
3. Results
3.1. Development of berry and seed
The growth curve of the berry, characterized by changes in width, had a doublesigmoid form (Fig. 1). The first development phase, a phase of rapid growth after
full bloom, lasted 38 days, and was followed by a period of decelerated growth
for 20 days. About 80% of the growth in berry width was observed in the first
36
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
Fig. 1. Changes in berry diameter and seed fresh weight of `Muscat Bailey A' grapes during
development and ripening. Vertical bars indicate the SE of the means (n 10). I, II and III indicate
the growth phase as follows: I, a period of rapid growth after anthesis; II, a period of decelerated
growth; and III, a second period of rapid growth during maturation.
phase. The onset of the third phase was at 58 days after full bloom (DAB), and the
duration of the third phase, a second period of increased growth, was 43 days.
Grapes were harvested at 101 DAB, when the berries contained over 18% soluble
solids. Seed development followed a different course from berry development.
The increase in seed fresh weight continued until 50 DAB, the highest growth rate
being from 20 to 30 DAB.
3.2. Changes in PA levels in pericarp
In all fractions, putrescine (Put), spermidine (Spd) and spermine (Spm) were
detected in the extract of pericarp (Fig. 2). Put was the predominant PA in all
fractions, and the levels of bound PA were highest during the development period.
In the free fraction, Put and Spd were highest at full bloom, thereafter gradually
decreasing during development phase I, with an increase again, especially of Put,
at 30 DAB. The PA conjugated increased at 30 DAB, and quickly decreased.
Bound PA exhibited changes similar to PA conjugates. Conjugated and bound Put
slightly increased in phase III. PA levels in all fractions, except for conjugated
and bound Put, were virtually constant, during development phases II and III. In
all fractions, the changes in Spm levels were negligible compared to those of Put
and Spd throughout the experiment.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
37
Fig. 2. Changes in polyamine levels in the pericarp of `Muscat Bailey A' grapes during
development and ripening. Data are means SE of three replicates.
38
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
Fig. 3. Changes in polyamine levels in seed of `Muscat Bailey A' grapes during development and
ripening. Data are means SE of three replicates.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
39
3.3. Changes in the levels of PA in seeds
In seeds, as in the pericarp, Put was the dominant PA in all fractions, with the
highest level found in bound PA (Fig. 3). However, the changes in PA levels
depended on the fraction. Free PA increased dramatically at 30 and 40 DAB.
Levels of free PA decreased rapidly at 50 DAB, reaching levels similar to those at
20 DAB. These levels showed little change thereafter up to harvest. Changes in
the level of Put conjugate followed a contrary course to that of free Put: a
decrease between days 20 and 30, increasing at 50 DAB to a level similar to that
of 20 DAB. The changes in the levels of Spd and Spm conjugates were less
obvious, in comparison with that of Put conjugate. The levels of bound PA were
almost constant during the first 40 DAB. Bound PA, like PA conjugates, increased
at 50 DAB and this increase was greatest in Put. From 50 DAB to harvest, bound
PA remained at higher levels.
4. Discussion
Polyamines (PA) identified in the pericarp and seed of grape were Put, Spd and
Spm, and, as with pepper (Serrano et al., 1995) and tomato (Casas et al., 1990),
Put was predominant in all fractions in both, the pericarp and seed throughout the
experiment. The concentration of PCA-insoluble bound PA was the highest, both
in the pericarp and seed. In the pericarp, free PA, especially Put and Spd, was
found at higher levels early in development, while they were lower in other
fractions during early development (Fig. 2). A high level of free PA in the early
phase of fruit development was also reported in fruits of other species (Biasi et
al., 1988; Ponappa and Miller, 1996), in which the direct involvement of PA in
cell division has been proposed. As shown in Fig. 1, the growth of the grape berry
is described by a double-sigmoid curve, indicating three development phases.
Phase I, a period of rapid growth after anthesis, is characterized by cell
proliferation followed by cell enlargement. The further growth found during
phase III resulted from cell enlargement at the outer wall parenchyma (Shiozaki
et al., 1997). In `Muscat Bailey A' grapes, cell division in almost all tissues of the
pericarp occurred from anthesis to 7 DAB (Nakagawa and Nanjo, 1966). High
levels of free Put and Spd during early development may, therefore, be associated
with cell proliferation in the pericarp.
The PA levels in all fractions increased 30 DAB, late in phase I (Fig. 2). The
increase in the levels of PA was greatest in Put in all fractions; in particular, PCAsoluble conjugated Put nearly doubled. These changes were similar to those in
cytokinin activity, which also reached a peak late in phase I in `Delaware' grape
pericarp (Inaba et al., 1976). Since the increase in PA levels in the pericarp late in
phase I was found in the seeded berries, but not in seedless ones (data not shown),
40
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
and occurred at the same time as free PA levels in the seeds increased, the
increase in PA in the pericarp 30 DAB may originate in the free PA synthesized in
the seeds. Also, the fact that an increase in PA 30 DAB was highest in the PCAsoluble conjugated fraction, with the PCA-insoluble bound fraction as second and
the free fraction the lowest, is an indication that the free PA exuded from the
seeds are immediately metabolized in the conjugated or bound forms in the
pericarp. Although the physiological role of the increase in PA late in phase I is
not clear, the increases may have little effect on pericarp development since the
increase in freeÐand supposedly activeÐPA (Smith, 1985) was negligible
compared to increases in the other fractions. Furthermore, the fact that cell
division had already ceased in almost all tissue of the pericarp, and that the rate of
cell enlargement is lower late in phase I (Nakagawa and Nanjo, 1966) tends to
support this hypothesis.
In the seeds, changes in the levels of PA were quite different from those in the
pericarp, being fraction dependent early in the development phase (Fig. 3). In the
seeds of `Muscat Alexandria' with a development period of 110 days, it was
reported that the maximum rate of mitosis in the outer integument occurs 20±25
days after bloom and that cell division in the endosperm is highest 35 days after
bloom (Pratt, 1971). The period of increasing free PA levels (between days 30 and
40) probably corresponds to increased cell division of the outer integument and
endosperm of the seeds of `Muscat Bailey A' grapes during a development period
of 101 days.
Although the role of PA in the development of the embryo is of interest, the
data presented in this study did not reveal changes in the levels of PA reflecting
embryo development. The grape embryo grows after seed development has
ceased, and reaches full size during phase III of berry development (Matsui,
1976). The PA levels in the seeds were almost constant during this period. To
elucidate the role of PA in the development of embryo, we would need to analyze
PA levels using isolated embryos.
References
Biasi, R., Bagni, N., Costa, G., 1988. Endogenous polyamines in apple and their relationship to fruit
set and fruit growth. Physiol. Plant 73, 201±205.
Casas, J.L., Acosta, M., Del Rio, J.A., Sabater, F., 1990. Ethylene evolution during ripening
of detached tomato fruit: its relation with polyamine metabolism. Plant Growth Regul. 9,
89±96.
Escribano, M.I., Merodio, C., 1994. The relevance of polyamine levels in cherimoya (Annona
cherimola Mill.) fruit ripening. J. Plant Physiol. 143, 207±212.
Evans, P.T., Malmberg, R.L., 1989. Do polyamines have roles in plant development?. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 40, 235±269.
Hasdai, D., Bar-Akiva, A., Goren, R., 1986. Chemical and morphological characteristics of
developing fruits from old clone vs. nucellar shamouti orange trees. J. Hort. Sci. 61, 389±395.
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Inaba, A., Ishida, M., Sobajima, Y., 1976. Changes in endogenous hormone concentrations during
berry development in relation to the ripening of Delaware grapes. J. Jpn. Soc. Hort. Sci. 45, 245±
252.
Kushad, M.M., Yelenosky, G., Knight, R., 1988. Interrelationship of polyamine and ethylene
biosynthesis during avocado fruit development and ripening. Plant Physiol. 87, 463±467.
Lavee, S., 1960. Effect of gibberellic acid on seeded grapes. Nature 185, 395.
Matsui, H., 1976. A study on the maturation of Delaware grape berry. Thesis. Osaka Prefecture
University, Osaka. (in Japanese).
Nakagawa, S., Nanjo, Y., 1966. Comparative morphology of the grape berry in three cultivars. J.
Jpn. Soc. Hort. Sci. 35, 29±38.
Nathan, R., Altman, A., Monselise, S.P., 1984. Changes in activity of polyamine biosynthetic
enzymes and in polyamine contents in developing fruit tissues of `Murcott' mandarin. Scientia
Hort. 22, 359±364.
Nitsch, J.P., 1970. Hormonal factors in growth and development. In: Hulme, A.C. (Ed.), The
Biochemistry of Fruits and their Products. Academic Press, London, New York. pp. 427±472.
Ponappa, T., Miller, A.R., 1996. Polyamines in normal and auxin-induced strawberry fruit
development. Physiol. Plant. 98, 447±454.
Pratt, C., 1971. Reproductive anatomy in cultivated grapesÐa review. Am. J. Enol. Vitic. 22, 92±
109.
Pyke, N.B., Alspach, P.A., 1986. Inter-relationships of fruit weight, seed number and seed weight in
kiwifruit. NZ Agri. Sci. 20, 153±156.
Scienza, A., Miravalle, R., Visai, C., Fregoni, M., 1978. Relationships between seed number,
gibberellin and abscisic acid levels and ripening in Cabernet Sauvignon grape berries. Vitis 17,
361±368.
Serrano, M., Martinez-Madrid, M.C., Riquelme, F., Romojaro, F., 1995. Endogenous levels of
polyamines and abscisic acid in pepper fruits during growth and ripening. Physiol. Plant. 95, 73±
76.
Shiozaki, S., Miyagawa, T., Ogata, T., Horiuchi, S., Kawase, K., 1997. Differences in cell
proliferation and enlargement between seeded and seedless grape berries induced parthenocarpically by gibberellin. J. Hort. Sci. 72, 705±712.
Smith, T.A., 1985. Polyamines. Ann. Rev. Plant. Physiol. 36, 117±143.
Smith, T.A., 1991. Chromatographic methods for the identification and quantitation of polyamines.
In: Slocum, R.D., Flores, H.E. (Eds.), Biochemistry and Physiology of Polyamines in Plants
1991. CRC press. Boca Raton, Ann Aorbor, London, pp. 229±242.
Toumadje, A., Richardson, D.G., 1988. Endogenous polyamine concentrations during development,
storage and ripening of pear fruits. Phytochem. 27, 335±338.
Endogenous polyamines in the pericarp and seed of
the grape berry during development and ripening
Shuji Shiozaki*, Tsuneo Ogata, Shosaku Horiuchi
College of Agriculture, Osaka Prefecture University, Gakuencho 1-1, Sakai Osaka 599-8531, Japan
Accepted 9 April 1999
Abstract
The levels of free, perchloric acid-soluble conjugated and perchloric acid-insoluble bound
polyamines were determined in pericarp and seeds of `Muscat Bailey A' grapes (Vitis labrusca
L. Vitis vinifera L.) during development and ripening. In both, the pericarp and seeds, putrescine
was the predominant polyamine in the three fractions, and the bound polyamine level was the
highest of the fractions. In the pericarp, the levels of free putrescine and spermidine were higher
during early development. In all fractions, all polyamines in the pericarp increased 30 days after full
bloom; the increase was greatest in conjugated polyamines, and least in free polyamines. These
increases coincided with an increase in the levels of free polyamines in the seeds. Polyamine levels
in all fractions were almost constant during ripening. In the seed, the levels of free polyamines
increased when the levels of conjugated polyamines decreased at 30 days after full bloom. The
levels of conjugated and bound polyamines increased 50 days after full bloom, with a decrease in
the free polyamine level. The inverse relation between the change in the levels of free polyamine
and of conjugated and/or bound polyamines was a peculiar feature to the seeds. # 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Grape berry; Grape seed; Polyamines; Putrescine; Spermidine; Spermine
1. Introduction
Polyamines (PA) are found in all organisms and are believed to be involved in
several physiological processes in higher plants, including morphogenesis,
* Corresponding author. Tel.: +81-722-549417; fax: +81-722-549417.
E-mail address: shiozaki@plant.osakafu-u.ac.jp (S. Shiozaki)
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 0 6 4 - 3
34
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
rooting, flowering and senescence (Evans and Malmberg, 1989). Although the
precise physiological role of PA in fruit development has not been established,
evidence for its involvement in the development and ripening of fruit arises from
the changes found in PA levels and metabolism in many fruits. These fruits are
divided into two groups according to the changes in PA levels. In the apple (Biasi
et al., 1988), avocado (Kushad et al., 1988), pear (Toumadje and Richardson,
1988), pepper (Serrano et al., 1995) and strawberry (Ponappa and Miller, 1996),
PA levels are high in the early phases of development and gradually decrease as
development progresses. In contrast, in mandarin (Nathan et al., 1984), orange
(Hasdai et al., 1986) and cherimoya (Escribano and Merodio, 1994), an increase
in PA levels is observed during ripening. The changes in PA levels may reflect
features of fruit growth and development in each fruit species.
In fruit, seeds are generally a metabolic center of phytohormones (Nitsch,
1970), so that the influence of seeds must be taken into account in discussions of
fruit development and ripening. The sizes of grapes and kiwifruits are closely
correlated with the numbers of seeds (Lavee, 1960; Pyke and Alspach, 1986). In
addition, Scienza et al. (1978) reported that the greater the number of seeds, the
higher the levels of gibberellin and abscisic acid in the pericarp of grape berries.
Since the phytohormones produced in seeds play a role in development of the
pericarp, PA produced in seeds may act in a similar manner.
Little evidence is currently available regarding the relationships between PA
levels in pericarp and seeds and the development of grape berry. In this study, we
analysed the levels of free, conjugated and bound PA in the pericarp and seeds of
grape berries during development and ripening. Relations between PA levels in
the pericarp and seeds and grape berry development and ripening are discussed.
2. Materials and methods
2.1. Plants
Three vines of four-year-old cv. `Muscat Bailey A' grapes planted in Osaka
Prefecture University were used. To study development of the berry, the width of
10 berries, selected randomly from five clusters and marked with thread, was
measured at 3±9-day intervals, from two days after full bloom (DAB) to harvest.
The fresh weight of 10 seeds taken from berries, which were randomly sampled
from five clusters, was determined at 10±20-day intervals from 20 DAB to
harvest.
Flowers and berries were randomly sampled from five clusters at 0, 10, 20, 30,
40, 50, 70, 90 DAB. On, and after, Day 20, seeds were taken from berries.
Replicate samples (500±700 mg) of flower, pericarp (including seeds in the 10
DAB sample) and seeds were frozen in liquid nitrogen, and stored at ÿ308C.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
35
2.2. Polyamine extraction
The extraction procedure of PA was essentially that of Smith (1991). Samples
were homogenized in cold 10% perchloric acid (PCA) (0.1 g tissue/ml PCA)
using a glass homogenizer, and the homogenate was maintained at 48C for
30 min. The extracts were centrifuged for 20 min at 12 500 g, and the supernatant fraction was used for the determination of free PA and PCA-soluble
conjugated PA. The pellet was used for the determination of PCA-insoluble
bound PA. It was washed in 5 ml of PCA, centrifuged for 20 min at 12 500 g,
then resuspended in the original volume of PCA by vortexing. The pellet
suspension and the original supernatant (0.2 ml each) were hydrolyzed for 18 h
with 0.2 ml of 12N HCl at 1108C in a reaction vial. The hydrolysate was
centrifuged and 0.1 ml aliquot of the supernatant was dried in vacuo at 608C, then
dissolved in 0.1 ml PCA. The soluble conjugated PA was estimated as the
concentration of PA in the hydrolysate of the original supernatant less that of the
free PA.
2.3. Dansylation of polyamines and HPLC analysis
The extracts were dansylated as described by Smith (1991). An aliquot (0.1 ml)
of the extract was added to 0.2 ml saturated sodium carbonate and 0.4 ml dansyl
chloride in acetone (7.5 mg/ml). The mixture was incubated at 608C for 30 min in
the dark. In order to eliminate excess dansyl chloride, 0.1 ml of proline (0.1 g/ml)
was added to the mixture which was incubated at room temperature for 15 min in
the dark. Dansylated PA were extracted with 0.5 ml toluene by vortexing for
1 min, and a 0.2 ml aliquot of toluene was dried. The derivatives were redissolved
in methanol and analyzed by reverse-phase HPLC with a fluorescence detector.
The excision and emission wavelengths were, respectively, 365 and 510 nm.
Samples were eluted from the reversed-phase HPLC column (4.6 mm 250 mm)
using a linear solvent gradient, from 60% methanol in pH 3.5 acetate buffer to
95% methanol, over 25 min, the latter for 10 min at a flow rate of 1 ml/min. Each
determination was performed in triplicate.
3. Results
3.1. Development of berry and seed
The growth curve of the berry, characterized by changes in width, had a doublesigmoid form (Fig. 1). The first development phase, a phase of rapid growth after
full bloom, lasted 38 days, and was followed by a period of decelerated growth
for 20 days. About 80% of the growth in berry width was observed in the first
36
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
Fig. 1. Changes in berry diameter and seed fresh weight of `Muscat Bailey A' grapes during
development and ripening. Vertical bars indicate the SE of the means (n 10). I, II and III indicate
the growth phase as follows: I, a period of rapid growth after anthesis; II, a period of decelerated
growth; and III, a second period of rapid growth during maturation.
phase. The onset of the third phase was at 58 days after full bloom (DAB), and the
duration of the third phase, a second period of increased growth, was 43 days.
Grapes were harvested at 101 DAB, when the berries contained over 18% soluble
solids. Seed development followed a different course from berry development.
The increase in seed fresh weight continued until 50 DAB, the highest growth rate
being from 20 to 30 DAB.
3.2. Changes in PA levels in pericarp
In all fractions, putrescine (Put), spermidine (Spd) and spermine (Spm) were
detected in the extract of pericarp (Fig. 2). Put was the predominant PA in all
fractions, and the levels of bound PA were highest during the development period.
In the free fraction, Put and Spd were highest at full bloom, thereafter gradually
decreasing during development phase I, with an increase again, especially of Put,
at 30 DAB. The PA conjugated increased at 30 DAB, and quickly decreased.
Bound PA exhibited changes similar to PA conjugates. Conjugated and bound Put
slightly increased in phase III. PA levels in all fractions, except for conjugated
and bound Put, were virtually constant, during development phases II and III. In
all fractions, the changes in Spm levels were negligible compared to those of Put
and Spd throughout the experiment.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
37
Fig. 2. Changes in polyamine levels in the pericarp of `Muscat Bailey A' grapes during
development and ripening. Data are means SE of three replicates.
38
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
Fig. 3. Changes in polyamine levels in seed of `Muscat Bailey A' grapes during development and
ripening. Data are means SE of three replicates.
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
39
3.3. Changes in the levels of PA in seeds
In seeds, as in the pericarp, Put was the dominant PA in all fractions, with the
highest level found in bound PA (Fig. 3). However, the changes in PA levels
depended on the fraction. Free PA increased dramatically at 30 and 40 DAB.
Levels of free PA decreased rapidly at 50 DAB, reaching levels similar to those at
20 DAB. These levels showed little change thereafter up to harvest. Changes in
the level of Put conjugate followed a contrary course to that of free Put: a
decrease between days 20 and 30, increasing at 50 DAB to a level similar to that
of 20 DAB. The changes in the levels of Spd and Spm conjugates were less
obvious, in comparison with that of Put conjugate. The levels of bound PA were
almost constant during the first 40 DAB. Bound PA, like PA conjugates, increased
at 50 DAB and this increase was greatest in Put. From 50 DAB to harvest, bound
PA remained at higher levels.
4. Discussion
Polyamines (PA) identified in the pericarp and seed of grape were Put, Spd and
Spm, and, as with pepper (Serrano et al., 1995) and tomato (Casas et al., 1990),
Put was predominant in all fractions in both, the pericarp and seed throughout the
experiment. The concentration of PCA-insoluble bound PA was the highest, both
in the pericarp and seed. In the pericarp, free PA, especially Put and Spd, was
found at higher levels early in development, while they were lower in other
fractions during early development (Fig. 2). A high level of free PA in the early
phase of fruit development was also reported in fruits of other species (Biasi et
al., 1988; Ponappa and Miller, 1996), in which the direct involvement of PA in
cell division has been proposed. As shown in Fig. 1, the growth of the grape berry
is described by a double-sigmoid curve, indicating three development phases.
Phase I, a period of rapid growth after anthesis, is characterized by cell
proliferation followed by cell enlargement. The further growth found during
phase III resulted from cell enlargement at the outer wall parenchyma (Shiozaki
et al., 1997). In `Muscat Bailey A' grapes, cell division in almost all tissues of the
pericarp occurred from anthesis to 7 DAB (Nakagawa and Nanjo, 1966). High
levels of free Put and Spd during early development may, therefore, be associated
with cell proliferation in the pericarp.
The PA levels in all fractions increased 30 DAB, late in phase I (Fig. 2). The
increase in the levels of PA was greatest in Put in all fractions; in particular, PCAsoluble conjugated Put nearly doubled. These changes were similar to those in
cytokinin activity, which also reached a peak late in phase I in `Delaware' grape
pericarp (Inaba et al., 1976). Since the increase in PA levels in the pericarp late in
phase I was found in the seeded berries, but not in seedless ones (data not shown),
40
S. Shiozaki et al. / Scientia Horticulturae 83 (2000) 33±41
and occurred at the same time as free PA levels in the seeds increased, the
increase in PA in the pericarp 30 DAB may originate in the free PA synthesized in
the seeds. Also, the fact that an increase in PA 30 DAB was highest in the PCAsoluble conjugated fraction, with the PCA-insoluble bound fraction as second and
the free fraction the lowest, is an indication that the free PA exuded from the
seeds are immediately metabolized in the conjugated or bound forms in the
pericarp. Although the physiological role of the increase in PA late in phase I is
not clear, the increases may have little effect on pericarp development since the
increase in freeÐand supposedly activeÐPA (Smith, 1985) was negligible
compared to increases in the other fractions. Furthermore, the fact that cell
division had already ceased in almost all tissue of the pericarp, and that the rate of
cell enlargement is lower late in phase I (Nakagawa and Nanjo, 1966) tends to
support this hypothesis.
In the seeds, changes in the levels of PA were quite different from those in the
pericarp, being fraction dependent early in the development phase (Fig. 3). In the
seeds of `Muscat Alexandria' with a development period of 110 days, it was
reported that the maximum rate of mitosis in the outer integument occurs 20±25
days after bloom and that cell division in the endosperm is highest 35 days after
bloom (Pratt, 1971). The period of increasing free PA levels (between days 30 and
40) probably corresponds to increased cell division of the outer integument and
endosperm of the seeds of `Muscat Bailey A' grapes during a development period
of 101 days.
Although the role of PA in the development of the embryo is of interest, the
data presented in this study did not reveal changes in the levels of PA reflecting
embryo development. The grape embryo grows after seed development has
ceased, and reaches full size during phase III of berry development (Matsui,
1976). The PA levels in the seeds were almost constant during this period. To
elucidate the role of PA in the development of embryo, we would need to analyze
PA levels using isolated embryos.
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