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Ed Etxeberria and Pedro Gonzalez

The mechanisms of sucrose uptake into the vacuole and sucrose efflux from the vacuole were studied using tonoplast vesicles from red beet at two distinct developmental stages. Vesicles from both developing and mobilizing hypocotyls (sucrose uptake and efflux, respectively) accumulated sucrose against a concentration gradient. However, higher rates and maximal levels of sucrose accumulation were obtained with tonoplast from developing hypocotyls. ATP-dependent sucrose efflux was more pronounced in vesicles from mobilizing hypocotyls. Despite the apparent overlapping, the data indicate that the physiological mechanisms for sucrose uptake and sucrose efflux are separated in time and governed by the developmental state of the cell. Chemical name used: adenosine 5′-triphosphate (ATP).

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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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Kenneth Marsh, Pedro Gonzalez, and Ed Echeverría

Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ΔpH. Although not able to function as a H+ pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H+ pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.

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Andreas Brune, Mathias Müller, Lincoln Taiz, Pedro Gonzalez, and Ed Etxeberria

Vacuolar acidification was investigated in `Palestine' sweet (Citrus limmetioides Tanaka) and `Persian' acid lime [(Citrus aurantifolia (Christm.) Swingle] (vacuolar pHs of 5.0 and 2.1, respectively) using tonoplast vesicles isolated from juice cells. The ATPase activity of tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3 -, but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3 - and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in tonoplast vesicles from sweet limes, the maximum ΔpH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg+2 after the maximal ΔpH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ΔpH was observed in vesicles from acid limes, indicating a less H+ permeable membrane. Vacuolar ATPases from both cultivars exhibited identical pH optima and showed similar Mg+2 dependence, but only the acid lime ATPase activity was inhibited by Ca+2. These data confirm that the vanadate-sensitive form of the V-ATPase found in lemon and acid limes is specific to hyperacidifying tissues rather than to citrus juice cells. Sweet lime vacuoles bear the classical V-ATPase also found in vegetative plant tissues.

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Ed Etxeberria, Pedro Gonzalez, and Javier Pozueta-Romero

To investigate the mechanisms of sucrose transport and its accumulation into `Murcott' mandarin (Citrus reticulata Blanco) fruit, developmental changes in determinants of sink strength such as sucrose metabolizing enzymes, and sucrose transport across both plasmalemma and tonoplast were analyzed. Concurrently with sucrose levels, sucrose synthase, sucrose phosphate synthase and sucrose phosphate phosphatase increased throughout fruit development. Plasmalemma and tonoplast vesicles isolated from fruit collected at different developmental stages were analyzed for their transport capabilities. Sucrose uptake into energized plasmalemma vesicles was abolished by gramicidin, which is in accordance with the presence of an active symport mechanism of sucrose transport from the apoplast into the cytosol. Unexpectedly, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. More importantly, however, and in conformity with recent findings showing the occurrence of an endocytic mechanism of ion uptake in maize (Zea mays L.) root cells, citrus (Citrus L.) juice cells were shown to incorporate membrane impermeable dyes into their vacuoles in the presence of sucrose. High definition confocal microscopy revealed the co-localization of membrane impermeable markers in cytoplasmic vesicles and the formation of vesicles at the plasmalemma. The data provide evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole bypassing both the plasmalemma and tonoplast.

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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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Dongmei Wei, Chao Gao, and Deyi Yuan

cells, degeneration of tapetal cells, polarization in microspores during the formation of large vacuoles, and pollen wall formation after releasing from tetrads. Anther cell differentiation during anther development is strictly regulated in accordance

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Fenghua Shi, Chun Sui, Yue Jin, Hao Huang, and Jianhe Wei

formation of the vacuole in which portions of the cytoplasm are sequestered. The latter is manifested by invagination of the tonoplast and the subsequent engulfment of cytoplasmic fragments into the cavity of a pre-existing vacuole ( Aubert et al., 1996

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Pedro Gonzalez, James P. Syvertsen, and Ed Etxeberria

mechanisms that contribute to Na + tolerance are the membrane-localized Na+ transporters at the tonoplast and the plasmalemma ( Tester and Davenport, 2003 ). Tonoplast-bound Na + /H + antiporters mediate the removal of Na+ into the vacuole. Overexpression

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Michele R. Warmund

fragmented vacuoles were present in nutritive cells. Like with other cynipid-induced galls, the nucleolus of some nutritive cells appeared vacuolated and organelles were often clustered near the nucleus ( Rey, 1976 ; Rohfritsch, 1971 ). Bronner (1980) also