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 viniferaL.) 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)
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.
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 super-natant 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 500g, 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 mm250 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
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.
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.
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 PCA-soluble conjugated fraction, with the PCA-inPCA-soluble 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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