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Shohei Yamaki and Migifumi Ino

A study was conducted to determine the distribution of sugars in vacuoles, cytoplasm, and free space in apples (Malus domestica Bork) picked at the immature and mature stage of maturity. The volumes of free space and air space were 13.4% and 14.5%, respectively, in immature fruit, and 14.6% and 25.6%, respectively, in mature fruit. The inner cellular volume (vacuole + cytoplasm) was 72% and 60% for immature and mature fruit, respectively. About 90% of each sugar (glucose, fructose, sucrose, and sorbitol) was found in the vacuole. The concentration of total sugar in the inner cell or free space was 326 or 128 mm each in immature fruit and 937 or 406 mm each in mature fruit. Permeability to sugars across the plasma membrane and tonoplast also increased with fruit maturation, 7- to 30-fold for the tonoplast and 4- to 5-fold for the plasma membrane in mature compared to immature fruit. Cells in immature fruit apparently enlarge through higher turgor pressure from sequestering of sugars into vacuoles, and cease to enlarge in mature fruit as the amount of sugar unloading into the fruit is reduced due to the accumulation of sugar in the free space or cytoplasm.

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Ofosu-Anim John and Shohei Yamaki

Using the compartmental analysis method, the distribution of sucrose, glucose, and fructose and their efflux from the free space, cytoplasm, and vacuole were determined in Nyoho strawberries (Fragaria ×ananassa Duch.) picked 25 or 35 days after pollination (DAP). At both stages, >70% of total sugar accumulated in the vacuole. Concentration of sugar in the free space increased from 167 mm in fruit at 25 DAP to 217 mm at 35 DAP, whereas that within the cell (cytoplasm + vacuole) increased from 233 to 352 mm. Permeability of the plasma membrane to sucrose, glucose, and fructose was higher than that of the tonoplast and, except for that of fructose, the permeability of the plasma membrane to sugars increased with fruit maturation. ABA at 10-5 m compared to 10-4 m restricted the release of all sugars from fruit discs and was due mainly to reduced efflux across the plasma membrane rather than the tonoplast. Thus ABA may stimulate the accumulation of sugars in fruit flesh by restricting their efflux. Chemical name used: abscisic acid (ABA).

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Takaya Moriguchi, Tetsuro Sanada and Shohei Yamaki

Sugar levels and composition were determined in developing `Hakuto' peach (Prunus persica Batsch var. vulgaris Maxim.) fruit. Glucose and fructose in nearly equal amounts were the predominant sugars detected during the early stage of development. Sucrose subsequently began to accumulate and was the predominant sugar in mature fruit. Sorbitol remained at a low level throughout development. The large increase in the amount of sucrose was accompanied by a rapid increase in sucrose synthase (EC 2.4.1.13) activity. Sucrose phosphate synthase (EC 2.4.1.14) was also detected in flesh extracts, but the activities were low throughout development. Acid invertase (EC 3.2.1.26) activity was highest in young fruit and declined with development. Activity, however, increased again at a later stage of development. Peach fruit contained appreciable sorbitol oxidase activity, while other sorbitol-related enzymes were barely detectable, suggesting that transported sorbitol was predominantly converted to glucose. These results suggest that the supply of glucose and fructose depends on acid invertase and sorbitol oxidase, and that accumulation of sucrose depends on-sucrose synthase.

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Akio Suzuki, Yoshinori Kanayama and Shohei Yamaki

The properties of sucrose synthase (SS) isozymes partially purified from immature fruit (SS I) of Japanese pear (Pyrus serotina Rehder var. culta Rehder) were different than those of mature fruit (SS II). A clear difference in elusion pattern during DEAE-cellulose chromatography was observed, although the apparent molecular weight of the native proteins extracted from both stages was 350 kD. The Km value of SS II for UDP was similar to that for UDP-glucose; while with SS I, the Km for UDP was lower than that for UDP-glucose. This suggests that SS II activity favors sucrose synthesis compared with SS I, which favors sucrose cleavage. The optimum pH for activity toward sucrose synthesis was 8.0 for SS II and 8.5 to 9.5 for SS I. SS II from mature fruit may be an isozyme of SS occurring during periods of rapid sucrose accumulation, while SS I from immature fruit is more similar to the typical SS which functions mainly toward sucrose cleavage in many plants.

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Yoshinori Kanayama, Hitoshi Mori, Hidemasa Imaseki and Shohei Yamaki

Sorbitol plays a important role in the translocation of photosynthate in apple. Sorbitol-6-phosphate dehydrogenase (S6PDH, is the key enzyme regulating sorbitol biosynthesis. The cloning of functional gene like S6PDH provides the potential to elucidate the mechanism of production and translocation of sugar in the Rosaceae family and to manipulate endogenous sorbitol production in horticultural crops.

Poly(A)+RNA was prepared from apple seedlings and cDNA library constructed in an expression vector was screened by the loquat-S6PDH antibody prepared by Hirai (Natl. Res. Inst. Veg. Ornam. Plants & Tea, Japan). The cloned cDNA contained an open reading frame of 930 base pairs encoding a sequence of 310 amino acids. Identification of the cDNA was accomplished by expression of active enzyme in Escherichia coli harboring the cDNA and by the presence of a partial amino acid sequence identical to that found in the purified enzyme. Northern blot analysis showed the expression of S6PDH gene in apple seedlings.

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Takuro Suyama, Kunio Yamada, Hitoshi Mori, Kiyotoshi Takeno and Shohei Yamaki

A cDNA library was constructed from poly(A)+RNA extracted from pollinated fruit of `PMR-142' cucumber (Cucumis sativus L.). Subtraction hybridization was made between the cDNAs and poly(A)+RNA from unpollinated fruit to isolate cDNA clones that corresponded to the genes preferentially expressed in the pollinated fruit. We isolated three cDNAs, which were 756, 826, and 998 nucleotides long and designated Csf1, Csf2, and Csf3, respectively. When fruit growth was triggered by pollination, auxin treatment and natural parthenocarpy, Csf2 was always expressed. Time course of expression of the Csf2 gene was nearly parallel to that of the fruit growth. Nucleotide sequences of the Csf cDNAs were fully determined. Homology of the deduced amino acid sequence for Csf1 showed 75% identity with a pea extensin. Only 37%, 33%, and 26% homology was found between Csf2 and bell pepper CaSn-2, tobacco FB7-4, and opium poppy gMLP15, respectively. The Csf3 sequence showed 68% identity with the large subunit of 60S ribosomal protein L3 of Arabidopsis thaliana.

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Hideaki Yamaguchi, Yoshinori Kanayama, Junichi Soejima and Shohei Yamaki

Seasonal changes in the amounts of the NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, 1.1.1.14) protein in developing apple (Malus pumila Mill var. domestica Schneid) fruit were determined by immunoblotting analysis. The amounts of the enzyme protein were very low in young fruit and rose as fruit matured. The weak correlation between enzyme protein and NAD-SDH activity and also the changes in NAD-SDH specific activity suggested that there could be posttranslational modification to the pre-existing enzyme or isoenzyme(s) of NAD-SDH. The changes in the amounts of NAD-SDH protein did not show the same pattern as those in relative growth rate, which is used to express sink activity, especially in young fruit. The role of NAD-SDH on sink activity in apple fruit, therefore, could not be explained simply by the amount and activity of the enzyme. In young fruit, it seems that enzymes other than NAD-SDH would be more directly related with fruit growth.

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Takaya Moriguchi, Kazuyuki Abe, Tetsuro Sanada and Shohei Yamaki

Soluble sugar content and activities of the sucrose-metabolizing enzymes sucrose synthase (SS) (EC 2.4.1.13), sucrose-phosphate synthase (SPS) (EC 2.4.1.14), and acid invertase (EC 2.4.1.26) were analyzed in the pericarp of fruit from pear cultivars that differed in their potential to accumulate sucrose to identify key enzymes involved in sucrose accumulation in Asian pears. The Japanese pear `Chojuro' [Pyrus pyrifolia (Burro. f.) Nakai] was characterized as a high-sucrose-accumulating type based on the analysis of mature fruit, while the Chinese pear `Yali' (P. bretschneideri Rehd.) was a low-sucrose-accumulating type throughout all developmental stages. The activity of SS and SPS in `Chojuro' increased during maturation concomitant with sucrose accumulation, whereas the activity of these enzymes in `Yali' did not increase during maturation. The activity of SS and SPS in the former were seven and four times, respectively, higher than those in the latter at the mature stage. Further, among 23 pear cultivars, SS activity was closely correlated with sucrose content, while SPS activity was weakly correlated. Soluble acid invertase activity in `Chojuro' and `Yali' decreased with fruit maturation, but the relationships between soluble invertase activity and sucrose content were not significant. The results indicate that SS and SPS are important determinants of sucrose accumulation in Asian pear fruit and that a decrease of soluble acid invertase activity is not absolutely required for sucrose accumulation.

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Motoko Iida, Nancy A. Bantog, Kunio Yamada, Katsuhiro Shiratake and Shohei Yamaki

The regulation of NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14) by sugar was investigated by using sliced tissues of japanese pear (Pyrus serotina Nakai cv. Kousui) fruit in order to determine its role in the mechanism of sugar accumulation in fruit tissue. The results of the activities and steady-state levels of the protein and mRNA indicate that NAD-SDH in japanese pear fruit is among the sugar-inducible genes. By preincubating the sliced tissues for 16 hours in a medium without sugar, NAD-SDH activity declined and reached a stable level that was maintained for up to 40 hours. The washing procedure also reduced the sugar concentration in the apoplast and cytosol of the sliced tissues to low concentrations and enabled them to be manipulated by exogenous applications of carbohydrate solutions. Incubation of tissues in 50 or 100 mm sorbitol for 8 hours led to enhanced expression of the NAD-SDH gene as determined by increased mRNA and protein levels and enhanced enzyme activity. The presence of 100 mm glucose, sucrose, or mannitol also gave significant stimulation on the levels of activity, protein, and mRNA of NAD-SDH compared with those of control tissues bathed in media in which the osmotic potential had been adjusted to that of the sugar solutions by adding polyethylene glycol. However, fructose was ineffective in stimulating NAD-SDH activities and the level of the protein was not enhanced but the level of mRNA was increased. Therefore, it is suggested that NAD-SDH gene transcription is enhanced by each sugar investigated, and fructose appears to be unique as it also influences NAD-SDH at a post-transcriptional level.

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Akira Tateishi, Kenji Nagashima, Francis M. Mathooko, Mercy W. Mwaniki, Yasutaka Kubo, Akitsugu Inaba, Shohei Yamaki and Hiroaki Inoue

Galactosidases are thought to play a key role in cell wall metabolism during fruit growth and ripening. In this study we cloned seven β-galactosidase (β-Gal) cDNAs from japanese pear fruit and designated them PpGAL2, PpGAL3, Pp-GAL4, PpGAL5, PpGAL6, PpGAL7, and PpGAL8, in addition to the previously described JP-GAL hereinafter termed PpGAL1. mRNA expression patterns of these clones were characterized throughout fruit growth and on-tree ripening, and in leaves and shoots in three japanese pear cultivars, `Housui', `Kousui', and `Niitaka'. The shared amino acid sequence identity among the eight japanese pear β-Gal (PpGAL) clones ranged from 50% to 60%. They all contained the putative active site containing consensus sequence pattern G-G-P-[LIVM](2)-x(2)-Q-X-E-N-E-[FY] belonging to glycoside hydrolase family 35. Expression of all the clones was both development- and tissue-specific. PpGAL1 and Pp-GAL4 were only expressed in the ripe fruit while PpGAL2 and PpGAL3 were expressed in both expanding and ripening fruit with their abundance being highest in the ripe fruit. The abundance of PpGAL5, PpGAL6, and PpGAL7 mRNAs was highest in expanding fruit but decreased drastically upon the onset of ripening. PpGAL8 was only detected in very young fruit (15 days after full bloom) and not in expanding and ripening fruit. These results indicate that in japanese pear fruit β-Gal is encoded by a multigene family whose members show distinct and overlapping expression during the various phases of fruit development. Some of the members are not only fruit-specific but also ripening-specific and, therefore, may play a crucial role in cell wall disassembly during japanese pear fruit softening.