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.
Shohei Yamaki and Migifumi Ino
Shohei Yamaki and Kazuko Ishikawa
Activities and roles of 4 sorbitol enzymes, sorbitol-6-P dehydrogenase, NAD+-dependent sorbitol dehydrogenase, NADP+-dependent sorbitol dehydrogenase and sorbitol oxidase, and acid invertase in apple (Malus domestica Borkh. ‘Jonnagold’) leaves and fruit were studied. Almost all of the soluble carbohydrates in leaves are present as sorbitol throughout the season. Sorbitol-6-P dehydrogenase had the highest activity among the enzymes, being high in young leaves and decreasing with age; whereas NAD+- and NADP+-dependent sorbitol dehydrogenases and sorbitol oxidase activities were barely detectable. Sorbitol was translocated from leaves to fruits where it was readily metabolized to other sugars, so the sorbitol concentration did not increase. NAD+-dependent sorbitol dehydrogenase that converts sorbitol to fructose had the highest activity of the 4 enzymes in developing fruits. Its activity rose in June, decreased in midseason, and increased again with fruit maturation. The fluctuation in enzyme activity corresponded to changes in fructose concentration. Sorbitol oxidase activity, which was about one-fifth that of NAD+-dependent sorbitol dehydrogenase, increased proportionately as fruits enlarged. Acid invertase activity was distinctly higher than sorbitol enzyme activities in both leaves and fruit, but its roles in sugar translocation and metabolism were not clearly established. The levels of sorbitol in stems and peduncles remained relatively constant during the season indicating that little metabolism occurred in the phloem during transit.
Sorbitol-6-phosphate dehydrogenase was found in the cotyledons of apple seedling (Malus domestica Borkh). A rise in enzyme activity coincided with sorbitol accumulation.
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).
Takaya Moriguchi and Shohei Yamaki
A convenient method of conserving grape (Vitis labrusca L. cv. Delaware; F. thunbergii; V. vinifera L. cv. Rizamat) was developed by culturing nodal segments in Murashige and Skoog’s medium, but at a lower than normal concentration of ammonium nitrate under standard tissue culture conditions consisting of a 16-hr photo-period at 28C. The cultures had a higher rate of survival after 262 to 290 days of storage under low ammonium nitrate condition than under low temperature (5 or 10C).
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.
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 220.127.116.11) activity. Sucrose phosphate synthase (EC 18.104.22.168) was also detected in flesh extracts, but the activities were low throughout development. Acid invertase (EC 22.214.171.124) 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.
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.
Hideaki Yamaguchi, Yoshinori Kanayama, Junichi Soejima, and Shohei Yamaki
Seasonal changes in the amounts of the NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, 126.96.36.199) 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.
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.