Tonoplast Vesicles Research Articles

Properties of enhanced tonoplast zinc transport in naturally selected zinc-tolerant silene vulgaris

It was demonstrated recently that isolated tonoplast vesicles derived from plants of a Zn-tolerant ecotype of Silene vulgaris accumulate more Zn than vesicles derived from a Zn-sensitive ecotype. We have now characterized the tonoplast-transport system that causes this uptake difference and demonstrated its genetic correlation to Zn tolerance using plant crosses. We conclude that the tonoplast Zn uptake system of the tolerant ecotype differs greatly in its characteristics from that of the sensitive one, with the most prominent differences being its insensitivity to protonophores and ortho-vanadate and its stimulation by Mg-GTP. These differences in characteristics are most likely due to the fact that Zn can be taken up by two or more parallel pathways, which are not present in the same proportions in both ecotypes. In both ecotypes, Zn is actively transported across the tonoplast (temperature coefficient > 1.6), most likely as a free ion, since citrate does not accumulate in vesicles. Most importantly, the uptake difference found using the ecotypes was also found between homozygous Zn-tolerant and Zn-sensitive F3 plants, proving the genetic correlation between increased tonoplast Zn transport and naturally selected Zn tolerance in S. vulgaris.

A comparison of Zn, Mn, Cd, and Ca transport mechanisms in oat root tonoplast vesicles

Oat root tonoplast vesicles were used to determine if tonoplast transport of the divalent cations Zn and Mn occurs via an antiport mechanism, like that described for Ca and Cd. Also, inhibitors reported to affect Ca transport were tested for their effects on Cd versus Ca transport and tonoplast ATPase activity. The ability of Ca, Cd, Zn, and Mn to alter the proton gradient was monitored using both the fluorescent probe acridine orange and C‐methylamine accumulation. After the proton gradient was established in MgATP‐energized vesicles, addition of Ca, Cd, and Zn to the reaction restored the fluorescence of acridine orange, indicating dissipation of the proton gradient. Fluorescence recovery was linearly correlated with metal concentration and followed the order Ca>Cd≧Zn. Addition of Mn did not restore the fluorescence of acridine orange. All four ions released C‐methylamine from MgATP‐energized vesicles in an ion‐concentration‐dependent manner, and with relative initial rates in the order of Ca>Cd>Zn>Mn. The observed ion‐concentration‐dependent release of protons from sealed vesicles suggests that Zn and Mn, like Ca and Cd, can be antiported into the plant vacuole. In an effort to assess whether Ca and Cd use the same carrier, we tested the effects of verapamil, Do‐Tea‐Br, nifedipine, ruthenium red, and LaCl2 on Ca versus Cd transport, and also on MgATPase activity. These compounds are shown to alter Ca transport in plants. Although some of the inhibitors had a negative effect on MgMAPase activity, the decrease in this activity did not account for the decrease in Ca or Cd transport observed in any case. Particularly verapamil had a much greater effect on Ca transport than Cd transport activity while not inhibiting ATPase substantially. Data presented provide evidence for Zn and Mn antiport activity in oat root tonoplast and show differences in responses of Ca and Cd antiport activities to several transport inhibitors.

Oligomeric state of the tonoplast proteins: Structure of the putative malate transporter in Catharanthus roseus

Vacuolar malate transport in Catharanthus roseus is probably mediated by a 37 kDa intrinsic tonoplast protein identified with a photolyzable malate analog. Antibodies raised against the protein inhibit malate uptake in isolated vacuoles. We report here the native molecular mass and the oligomeric state of the putative malate transporter which were determined from two‐dimensional native electrophoresis. In its first dimension, the electrophoresis used a charge shift method developed for isolating native membrane protein complexes from purified tonoplast vesicles. In combination with a second dimension of sodium dodecylsulfate electrophoresis, it enables the determination of the oligomeric state and subunit composition of non‐dissociated complexes. In such analyses, most of the tonoplast proteins of Catharanthus roseus appear to have a complex structure. In native gels (first dimension), both the photoprobe and the antibodies recognized a 160 kDa protein. The photolabelling characteristics correlate well with the main features of malate transport activity. The 160 kDa protein, when analyzed in the second dimension, contained the 37 kDa polypeptide as a subunit. In addition, cross‐linking with dimethyl suberimidate (DMS) in the intact tonoplast vesicles resulted in the disappearance of the 37 kDa monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer, i.e. 73 and 160 kDa. These results, taken together, suggest that the putative malate transporter exists in the tonoplast as a tetramer.

Changes of H+ pumps of tonoplast vesicle from wheat roots in vivo and in vitro under aluminum treatment and effect of calcium

Wheat seedlings were treated with 0.1 mM Al for 7 days. Tonoplast vesicles were isolated from apical segments of roots and activities of H+‐ATPase and H+‐PPase measured. Compared with the control, 0.1 mM Al increased H+‐ATPase activity and decreased H+‐PPase activity. High external supply of calcium (Ca) (5 mM) diminished the extent of the stimulation of H+‐ATPase activity and alleviated the reduction of H+‐PPase activity under Al treatment. However, 0.1 mM Al treatment in vitro resulted in the inhibition of H+‐ATPase activity with the decrease of Vmax and Km for ATP. In vitro treatment with 0.1 mM Al also decreased the H+‐PPase activity, but increased the Km for PPi.

Citrate uptake into tonoplast vesicles from acid lime (Citrus aurantifolia) juice cells.

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 µM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity.

Effects of calcium on lipid composition and function of plasma membrane and tonoplast vesicles isolated from roots of barley seedlings under salt stress

The effects of salt stress(200 mmol L‐1) and calcium (Ca) on the lipid composition and function of plasma membrane and tonoplast vesicles isolated from the roots of barley seedlings (Hordeum vulgare L. Jian 4) have been investigated. Nine days of exposure to 200 mmol L‐1 NaCl resulted in a significant increase in the electrolyte leakage and Na+/K+ ratio in the roots, sheaths, and leaves of barley. The index ofunsaturated fatty acids (IUFA) in plasma membrane (PM) and tonoplast (TP) vesicles, and the levels of galactolipid in TP increased (p<0.01). The contents of phospholipids in PM decreased (p<0.05). In contrast to salt treatment, supplement of Ca2+(17.5 mmol L‐1) obviously increased the phospholipids content, and significantly decreased the levels of galactolipids and IUFA in PM and TP vesicles. The results showed that the alleviating effect of Ca2+ on NaCl induced injury in barley seedlings may be related to the changes of membrane lipid composition including phospholipids, galactolipids, and their fatty acids.

Modulation of the vacuolar H+-ATPase by adenylates as basis for the transient CO2-dependent acidification of the leaf vacuole upon illumination

Using tonoplast vesicles, we have investigated the activity of the vacuolar H+-ATPase which is the dominant proton pump at the tonoplast of mesophyll cells. Bafilomycin-sensitive ATP hydrolysis or acidification of tonoplast vesicles in the presence of ATP were measured at varying ATP, ADP and Pi concentrations, and in the presence of oxidized or reduced glutathione. Increased ATP/ADP ratios as reported for the extrachloroplast cytoplasm during the induction phase of photosynthesis at high or low CO2 (P. Gardeström, Biochim. Biophys. Acta 1183 (1993) 327–332) increased the activity of the V-ATPase in simulation experiments with vesicles. Depending on reported subsequent decreases in cytoplasmic ATP/ADP ratios in the presence of high or low CO2, the ATPase activity of tonoplast vesicles changed in simulation experiments to lower values. More than 10 mM phosphate was required to decrease the ATPase activity in vesicles significantly at ATP/ADP ratios of 3 or higher, indicating that ATPase activity is controlled more by ratios of ATP to ADP than by phosphorylation potentials (ATP)/(ADP)(Pi). Oxidized glutathione was inhibitory. The results permit interpretation of the observation that on illumination of previously darkened leaves the pH of the vacuoles of mesophyll cells decreases indicating energized transport of protons across the tonoplast into acidic vacuoles, and that the extent of vacuolar acidification depends on the CO2 concentration of the surrounding air (Z.-H. Yin, S. Neimanis, U. Heber, Planta 182 (1990) 253–261). We conclude that short term control of tonoplast ATPase activity in leaves during dark/light transients can essentially be understood on the basis of reported changes in cytoplasmic ATP/ADP ratios, with a possible participation of redox modulation.

Effects of salt stress on H +-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots

It has previously been shown that mild and severe salt-stress increased both ATP- and PP i-dependent H + transport and induced a Na +/H + exchange activity in tonoplast vesicles from sunflower seedling roots. In this study, the vanadate-sensitive H +-ATPase activity was characterized in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to 0, 75 or 150 mM NaCl. Blots immunoassayed with an Arabidopsis thaliana plasma membrane ATPase polyclonal antibody revealed the existence of a band of about 100 kDa, which was highly enriched in the 32/43% sucrose interface. ATP hydrolyzing activity in this fraction was mostly inhibited by vanadate, and scarcely by azide, oligomycin, nitrate and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Properties of vanadate-sensitive ATPase activity, such as inhibitor sensitivity, pH optimum, substrate specificity, ion effects and kinetic data were consistent with those of other plasma membrane ATPases. Although the pH profile, ion dependence, apparent K m and the amount of antigenic protein were unaffected by salt-stress, the V max of the vanadate-sensitive ATPase activity (measured in the presence of detergent) was reduced as a function of salt treatments. Likewise, while mild and severe salt-stress reduced the basal ATP hydrolyzing activity (measured in the absence of detergent) about a 35 and 55%, respectively, the ATP-dependent H + transport activity sensitive to vanadate remained unchanged. As a consequence, the ratio of H + pumping to the basal phosphohydrolase activity was proportionally increased. These results and those previously reported at the sunflower tonoplast suggest that the activity of H + pumps under salt stress can form part of a tolerance mechanism in sunflower roots, which could regulate the ion fluxes (Na + and Cl −) across the tonoplast and plasma membrane.

Comparison of Mg 2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

The characteristics of the Mg 2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 m M KH 2PO 4–K 2HPO 4, 5 m M KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 m M sucrose, 5 m M K 2HPO 4 and 4 m M KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E 1E 2–ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg 2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.

Proton pumps and anion transport in Vitis vinifera: The inorganic pyrophosphatase plays a predominant role in the energization of the tonoplast

In order to gain insight into vacuolar changes that are associated with fruit specialization regarding the accumulation of organic acids, the properties of tonoplast vesicles extracted from grape cell culture and ripe berries were compared. The two primary pumps, PPase (Pyrophosphatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture tonoplast vesicles, exhibited classical behaviour for known inhibitors and activators. However, the 3-fold higher activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolytic activities of both pumps were stimulated by temperatures up to 65 °C. Anion transport was studied by measuring simultaneously proton entry and membrane potential on the two types of tonoplast vesicles. Maximal acidification rates could be observed upon simultaneous energization by ATP and PPi, and were abolished by imidodiphosphate and bafilomycin, while vanadate had little effect. Comparison of the sequence of transport rates observed with mineral and organic anions on fruit or cultured cell tonoplast vesicles suggested that the fruit vacuole was more specialized in the transport of organic anions than the cultured cells. The transport of malate and tartrate was completely inhibited by 4,4′-diisothio-cyanato-stilbene-2,2′disulphonic acid while chloride transport was less affected. Km values for malate and tartrate transports were similar for vesicles extracted from cultured cell and fruits, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the vacuolar uptake of the two major acids of grape berries.

Reversibility of H+-ATPase and H+-pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to 32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.

Inhibition of vacuolar proton pumps by ethanol impairs grape berry compartmentation

Tonoplast vesicles were extracted from mature grape berries in order to study the effect of ethanol on their vacuolar proton pumps, vacuolar ATPase (V‐ATPase) and inorganic pyrophosphatase (PPase). Ethanol significantly inhibited their functioning; PPase was more sensitive than V‐ATPase. The effectiveness of alcohols with different carbon‐chain length indicated that a localised modification of the lipidic environment of the pump may be involved in this phenomenon. However, ethanol did not seem to significantly increase passive proton permeability of tonoplast vesicles. The inhibition of the hydrolytic activity of the inorganic pyrophosphatase PPase led to a decrease in the proton pumping rate into the vacuole and consequently in the generated pH gradient. PPase inhibition by ethanol was not compensated by an activation of the less sensitive V‐ATPase when both pumps operated simultaneously. The response of proton pumps to ethanol would be expected to impair cellular compartmentation during anaerobic metabolism. This phenomenon would explain the cell death in grape berries during ‘carbonic maceration’ or ‘vinification beaujolaise’.

γ-Irradiation damage to the tonoplast in cultured spinach cells

The effects of γ-irradiation on the tonoplast of cultured spinach cells were investigated using purified tonoplast vesicles. The activities of the membrane-bound enzymes, i.e. ATPase and pyrophosphatase (PPase), were considerably reduced when the isolated vesicles were exposed to radiation (1 and 5 kGy), but this reduction was alleviated in the tonoplast irradiated in vivo. In the presence of an extract prepared from non-irradiated cells, the decrease of the enzyme activities in the irradiated vesicles was reduced. Similar alleviation effects were also observed with regards to fatty acid residues of membrane lipids as well as with SDS–PAGE detectable polypeptides of the membrane. From these results, we conclude that the tonoplast in vivo was protected from radiation at least partly through the action of certain metabolic product(s). Other noticeable features clarified in this investigation are: (1) ATPase is more susceptible to radiation than PPase, (2) unsaturated fatty acids in membrane lipids were markedly decreased, in contrast to unaltered saturated acids, (3) membrane proteins seem to be more susceptible to radiation as compared to membrane lipids.

H+‐atpase and H+‐transport activities in tonoplast vesicles from barley roots under salt stress and influence of calcium and abscisic acid1

Seedlings of two barley cultivars differing in NaCl sensitivity were treated with low (100 mM) or high (400 mM) concentration of NaCl for 6 days. Tonoplast vesicles were prepared from roots, and H+‐ATPase and H+‐transport activities associated with tonoplast were assayed. Both H+‐ATPase and H+‐transport activities in the two cultivars were increased at 100 mM NaCl. These activities also increased in the salt‐tolerant cultivar at 400 mM NaCl, but in salt‐sensitive cultivar were decreased. In vivo treatment with 10 mM Ca2+ stimulated H+‐ATPase and H+‐transport activities at two levels of NaCl, however, treatment with 105M (±) abscisic acid (ABA) inhibited these activities. From these results we propose that the increase of the vacuolar H+ pumps in barley roots reflects an adaptation to salt stress. The stimulation of HVATPase and H+‐transport activities by calcium (Ca) depends mainly on its effect in maintaining stability of membrane under salt stress.

Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots.

The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles, but relative rates of accumulation/association of metals with vesicles were Ca > Cd >> Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in oat roots.

Na+/H+ Antiporter in Tonoplast Vesicles from Rice Roots

The N a + / H + antiporter in vacuolar membranes transports Na + from the cytoplasm to vacuoles using a pH gradient generated by proton pumps; it is considered to be related to salinity tolerance. Rice (Oryza sativa L.) is a saltsensitive crop whose vacuolar antiporter is unknown. The vacuolar pH of rice roots, determined by P-nuclear magnetic resonance (NMR), increased from 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transport of protons into the tonoplast vesicles from rice roots was fluorometrically measured. Efflux of protons was accelerated by the addition of Na + . Furthermore, the influx of Na into the tonoplast vesicles was accelerated by a pH gradient generated by proton-translocating adenosine 5-triphosphatase (H-ATPase) and proton-translocating inorganic pyrophosphatase (H-PPase). We concluded that this N a + / H + antiporter functioned as a Na + transporter in the vacuolar membranes. The antiporter had a Km of 10 mM for Na + and was competitively inhibited by amiloride and its analogues. The K, values for 5-(A^-methyl-A^-isobutyl)-amiloride (MIA), 5-(/V-ethyl-./V-isopropyI)-aniiloride (EIPA), and 5-(iV,jV-hexamethylene)-amiloride (HMA) were 2.2, 5.9, and 2.9 ftM, respectively. Unlike barley, a salt-tolerant crop, NaCl treatment did not activate the antiporter in rice roots. The amount of antiporter in the vacuolar membranes may be one of the most important factors determining salt tolerance.

Evidence for an important role of the tonoplast in the mechanism of naturally selected zinc tolerance in Silene vulgaris

Summary Compartmentalization of zinc in the vacuole has been proposed to explain enhanced zinc tolerance in higher plants. In order to determine whether the vacuolar membrane is involved in differential zinc tolerance, tonoplast vesicles were isolated from roots of zinc tolerant and sensitive Silene vulgaris. In vitro transport studies with K + /nigericin energized tonoplast vesicles showed that zinc (supplied as zinc citrate) was transported across the tonoplast only in the presence of MgATP. Zinc transport was about 2.5 times higher in tonoplast vesicles derived from zinc tolerant Silene vulgaris compared with those derived from sensitive Silene vulgaris at high zinc concentration (0.8 mmol/L). At low zinc concentrations (0.1–0.3 mmol/L) there was no significant difference in zinc transport between both types of plants. The difference in zinc transport at high external zinc concentrations could be due to an additional transport system with lower zinc affinity that may operate at higher zinc concentrations. Overall, these data strongly suggest that the tonoplast plays an important role in naturally selected zinc tolerance.

Characterization of proton and sugar transport at the tonoplast of sweet lime (Citrus limmetioides) juice cells

Citrus fruits accumulate high levels of sucrose and hexoses, although most photoas‐similates arrive in the form of sucrose. In sweet limes, faster rates of sugar accumulation take place early in development when sucrose catabolic enzymes are most active. The present investigation was aimed at providing information on the mechanisms of sucrose (and hexose) uptake into the vacuole of cells containing high levels of sucrose hydrolytic activity. Tonoplast vesicles of high purity were isolated in a discontinuous sucrose gradient. The vesicles were capable of forming a pH gradient in the presence of ATP. Both bafilomycin and NO3− (but not vanadate) inhibited ATP hydrolysis and prevented the formation of the pH gradient, confirming the tonoplast origin. Energized vesicles (either by addition of ATP or by artificial pH gradient) did not accumulate sucrose or hexoses against a concentration gradient. In the presence of either sucrose or hexoses, the established ΔpH; was not disrupted as was the case with tonoplast vesicles from red beet hypocotyl. Therefore, a sucrose/H+ (hexose) antiport may not be the mechanism of sucrose and hexose transport into the vacuoles of sweet lime juice cells. The data indicated that sucrose uptake into vacuoles of sweet lime occurs by facilitated diffusion. Hexoses originate from the hydrolytic action of acid invertase on sucrose within the vacuole, and by the action of cytosolic sucrose synthase.

Characterization of Water Channels in Wheat Root Membrane Vesicles.

The functional significance of water channels in wheat (Triticum aestivum L.) root membranes was assessed using light scattering to measure vesicle shrinking in response to osmotic gradients rapidly imposed in a stopped flow apparatus. Vesicles were obtained from both a plasma membrane fraction and a plasma membrane-depleted endomembrane fraction including tonoplast vesicles. Osmotic water permeability (Pos) in the endomembrane fraction was high (Pos= 86.0 [mu]m s-1) with a low activation energy (EA= 23.32 kJ mol-1 [plus or minus] 3.88 SE), and was inhibited by mercurials (K1= 40 [mu]M HgCl2, where K1 is the inhibition constant for half-maximal inhibition), suggesting participation of water channels. A high ratio of osmotic to diffusional permeability (Pd) (using D2O as a tracer, Pos/Pd = 7 [plus or minus] 0.5 SE) also supported this view. For the endomembrane fraction there was a marked decrease in Pos with increasing osmotic gradient that was not observed in the plasma membrane fraction. Osmotic water permeability in the plasma membrane fraction was lower (Pos= 12.5 [mu]m s-1) with a high activation energy (EA= 48.07 kJ mol-1 [plus or minus] 3.63 SE) and no mercury inhibition. Nevertheless, Pos/Pd was found to be substantially higher than one (Pos= 3 [plus or minus] 0.2 SE), indicating that water channels mediated water flow in this fraction, too. Possible distortion of the Pos/Pd value by unstirred layer effects was shown to be unlikely.