Solute Compartmentation Research Articles

Hierarchical Structures, with Submillimeter Patterns, Micrometer Wrinkles, and Nanoscale Decorations, Suppress Biofouling and Enable Rapid Droplet Digitization.

Liquid repellant surfaces have been shown to play a vital role for eliminating thrombosis on medical devices, minimizing blood contamination on common surfaces as well as preventing non-specific adhesion. Herein, an all solution-based, easily scalable method for producing liquid repellant flexible films, fabricated through nanoparticle deposition and heat-induced thin film wrinkling that suppress blood adhesion, and clot formation is reported. Furthermore, superhydrophobic and hydrophilic surfaces are combined onto the same substrate using a facile streamlined process. The patterned superhydrophobic/hydrophilic surfaces show selective digitization of droplets from various solutions with a single solution dipping step, which provides a route for rapid compartmentalization of solutions into virtual wells needed for high-throughput assays. This rapid solution digitization approach is demonstrated for detection of Interleukin 6. The developed liquid repellant surfaces are expected to find a wide range of applications in high-throughput assays and blood contacting medical devices.

Efficient use of energy in anoxia‐tolerant plants with focus on germinating rice seedlings

Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Tolerance of extreme salinity in two stem-succulent halophytes (Tecticornia species).

Communities of Tecticornia on the margins of ephemeral salt lakes in Australia often exhibit species zonation, such as at Hannan Lake (Western Australia) where Tecticornia indica subsp. bidens (Nees) K.A.Sheph. and Paul G.Wilson occupies the less saline dune habitat on lake margins and Tecticornia pergranulata (J.M.Black) K.A.Sheph. and Paul G.Wilson subsp. pergranulata occupies both the dunes and the more saline and moist lake playa. Here we tested the hypothesis that these two species differ in tolerance to extreme salinity. Plants were grown in drained sand cultures with treatments of 10-2000mM NaCl for 85 days. Both species were highly salt tolerant, maintaining growth at treatments of up to 2000mM NaCl, although the death of two replicates of T. indica at 2000mM NaCl suggests this salinity is close to the species tolerance limit. Both Tecticornia species maintained a favourable gradient in tissue water potential via osmotic adjustment as external salinity increased, also with reduced tissue water content at very high external salinity. Regulated accumulation of Na+ and Cl-, maintenance of net K+ to Na+ selectivity, high tissue concentrations of glycinebetaine and presumed cellular solute compartmentation, would have contributed to salt tolerance. The growth rate of T. pergranulata was 11-29% higher than T. indica suggesting, in addition to these moderate differences in salinity tolerance, other factors are likely to contribute to species zonation at salt lakes. The higher water use efficiency of the C4 T. indica compared with the C3 T. pergranulata may provide an advantage in the drier dune habitat on salt lake margins. An additional experiment confirmed the hypothesis that survival of T. pergranulata seedlings is enhanced by the duration of reduced salinity after germination, as would occur following significant rainfall, as older seedlings maintained higher growth rates during subsequent increases in salinity.

Architecture of vasa recta in the renal inner medulla of the desert rodent Dipodomys merriami: potential impact on the urine concentrating mechanism

We hypothesize that the inner medulla of the kangaroo rat Dipodomys merriami, a desert rodent that concentrates its urine to over 6,000 mosmol/kg H(2)O, provides unique examples of architectural features necessary for production of highly concentrated urine. To investigate this architecture, inner medullary vascular segments in the outer inner medulla were assessed with immunofluorescence and digital reconstructions from tissue sections. Descending vasa recta (DVR) expressing the urea transporter UT-B and the water channel aquaporin 1 lie at the periphery of groups of collecting ducts (CDs) that coalesce in their descent through the inner medulla. Ascending vasa recta (AVR) lie inside and outside groups of CDs. DVR peel away from vascular bundles at a uniform rate as they descend the inner medulla, and feed into networks of AVR that are associated with organized clusters of CDs. These AVR form interstitial nodal spaces, with each space composed of a single CD, two AVR, and one or more ascending thin limbs or prebend segments, an architecture that may lead to solute compartmentation and fluid fluxes essential to the urine concentrating mechanism. Although we have identified several apparent differences, the tubulovascular architecture of the kangaroo rat inner medulla is remarkably similar to that of the Munich Wistar rat at the level of our analyses. More detailed studies are required for identifying interspecies functional differences.

Salt Tolerance in the HalophyteHalosarcia pergranulatasubsp.pergranulata

Halosarcia pergranulata(P. G. Wilson) subsp.pergranulatais a member of the Salicornioideae and is native to Australia. Salt tolerance inH. pergranulatasubsp.pergranulatawas assessed by growing plants for 83 d at seven NaCl concentrations from 10 to 800 mol m−3. Shoot biomass was greatest for plants grown at 10 to 200 mol m−3NaCl, while at salinities of 300 mol m−3or higher it was inhibited. There was little increase in succulence in response to NaCl, and it even declined at the highest salinities. The K+:Na+molar ratio in succulent shoot tissues decreased from 0.30:1 in plants grown at 10 mol m−3NaCl to 0.02:1 in plants at 600 mol m−3, due to a three-fold increase in tissue Na+concentration and a five-fold decline in tissue K+. The osmotic potential of sap (πsap) in the expanding shoot tissues remained −1.7 to −2.5 MPa below the πof the root medium. Na+plus Cl−contributed 56 to 80% of the πsapin plants grown at 10 to 800 mol m−3NaCl. These findings show thatH. pergranulatasubsp.pergranulatahas a high degree of NaCl tolerance, and that studies on solute compartmentation at the cellular level are required to elucidate the mechanisms by which this species tolerates very low tissue K+:Na+ratios.

The intercellular distribution of vacuolar solutes in the epidermis and mesophyll of barley leaves changes in response to NaCl

Concentrations of inorganic and organic solutes were measured in sap extracted from individual mesophyll and epidermal cells of the third leaf of barley. During the development of the third leaf plants were grown in various salt solutions (NaCl; 2, 50, 100, and 150 mM, KCI; 100 mM or KNO3; 100 mM). Leaves were analysed 2–4 d after full expansion. Cell-sap was extracted using a modified pressure probe and analysed for its osmolality, concentrations of P, Na+ K+ Ca2+, and Cl− and, in some cases, of nitrate, hexoses and total amino acids. Salt treatment caused differential changes in the concentrations of solutes in mesophyll and epidermal cells, but did not affect the basic pattern of solute compartmentation between these tissues. Calcium was found at osmotically significant concentrations only in the epidermis, whereas P and organic solutes were almost exclusively found in the mesophyll. Chloride and Na+ accumulated preferentially in the epidermis, although mesophyll concentrations also increased considerably. At 150 mM external NaCl, mesophyll cells contained 302 mM Na and 167 mM Cl−, compared to 29 mM Na+ and 16 mM Cl− in the control. Mesophyll Cl− levels were even higher in the 100 mM KCl treatment (216 mM) where mesophyll and epidermal K+ accumulated to 424 and 491 mM, respectively. These huge increases in mesophyll Na+ Cl− and K+ were not associated with a breakdown in leaf performance since net rates of photosynthesis decreased only by less than 20%. Under control (2 mM NaCl) conditions, solutes followed patterned gradients between the various epidermal cell types. The extent of these gradients changed with leaf age. During 50 mM NaCl treatment, gradients in Cl−, nitrate and malate concentrations progressively disappeared, with malate concentrations approaching zero. Potassium and Na+ exhibited altered distribution profiles, whereas Ca2+ distribution was unaffected. NaCl-dependent increases in osmolalities differed between cells. Exposure of plants to 150 mM NaCl caused qualitatively similar changes in both epidermal solute and osmolality profiles, although absolute values differed from those at 50 mM NaCl. In particular, epidermal Cl− and Na+ increased to about 500 mM and K+ disappeared (≪5 mM) from the vacuole of certain epidermal cell types completely.

Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. II. Inter‐ and intracellular solute compartmentation in leaflets

ABSTRACTPea plants (Pisum sativum L. cv. ‘Kleine Rheinländerin’) grown on ammonium or nitrate as the sole nitrogen source were treated with 50 mol m−3 NaCl. Four days after salt addition, ammonium‐grown plants developed the first visible damage symptoms (wilting of leaflets, starting from the margins). Salt‐treated, nitrate‐grown plants were not affected during the experimental period. In order to obtain a better understanding of this differential salt sensitivity, we investigated the inter‐ and intracellular ion compartmentation of leaflets under both nutritional conditions by analysing ion concentrations in the apoplastic space, in chloroplasts and in protoplasts. When the leaves of nitrate‐ and ammonium‐grown plants had attained similar sodium and chloride contents (after different times of exposure to salinity), the latter had a considerably lower chloroplastic chloride (and also sulphate) concentration. The results suggest that the intracellular compartmentation capacity of ammonium‐grown plants is considerably lower than that of nitrate‐grown plants. Ion toxicity appeared to initiate breakdown of metabolism in parts of the mesophyll tissue of ammonium‐grown plants, causing an abrupt release of solutes into the apoplast, which coincided with the appearance of visible damage. Although the ammonium concentrations in leaves increased dramatically in the later phases of damage development, they were too low to cause the collapse of electrochemical gradients at the time at which damage became visible. Thus, the reason for a lower compartmentation capacity under ammonium nutrition remains as yet unclear.

The chaotropic anions thiocyanate and nitrate inhibit the electric current through the tonoplast ATPase of isolated vacuoles from suspension cells of Chenopodium rubrum

Using the whole‐vacuolar mode of the patch clamp technique, we studied the effect of the chaotropic anions thiocyanate and nitrate on the electric currents generated by the proton pumping tonoplast ATPase and pyrophosphatase (PPiase), respectively, in vacuoles from suspension cells of Chenopodium rubrum L. Addition of KNO3 (150–250 mM) or KSCN (70–150 mM), and ATP (5 mM, obligatory) irreversibly inhibited the subsequent electric current through the tonoplast ATPase driven by 1 mM ATP, whereas PPiase‐activity by 50 μM PPi remained unaffected. The kinetics of inhibition, indicative of ATPase disintegration by the chaotropic anions, follows a single exponential (τ= 3.44 min). However, apparent ATPase disintegration did not measurably increase the tonoplast conductance. We conclude that, by contrast to organellar F‐ATPases, upon disintegration the transmembrane proteolipid of the V‐ATPase does not act as a proton conductor which, in the presence of chaotropic anions, like chloride or nitrate, would severely perturb solute compartmentation in the plant cell.

Water Stress and the Water Relations of Seed Development: A Critical Review

Water availability plays a major role in the regulation of seed development. Experimental evidence on the water relations of seed development is reviewed, with emphasis on the measured water potential values of well‐watered and water‐stressed seed, fruit, and leaf tissues. The hypothesis of hydraulic isolation of developing seeds from the water relations of the maternal plant, proposed on the basis of the maintenance of measured water potential (ψw) differences between these tissues, is examined and found to be problematic. Apparent ψw gradients have also been recorded for seeds developing within fleshy fruits or in vitro on defined medium, where hydraulic barriers would not be present. Current models of phloem transport to seeds are based on a turgor homeostatic mechanism in the unloading tissues that maintains high apoplastic solute concentrations to lower phloem turgor and promote import. The combination of turgor (or water potential) boo meostasis and active regulation of solute compartmentation in seed tissues may contribute to the apparent constancy of seed ψw when measured by thermocouple psychrometry, which requires long equilibration times after excision. The delivery of excess water to developing seeds via the phloem and its recirculation to the mother plant also present problems for the hydraulic resistance hypothesis and for the retention of apoplastic solutes in the unloading zone. It is hypothesized that modfied cell walls acting as semipermeable apoplastic membranes may retain solutes within the unloading tissues, while allowing the return flow of water to the parental xylem. The water relations of developing seeds are determined primarily by phloem transport, whether under well‐watered or water‐stressed conditions, so future studies should attempt to resolve the discrepancies between measured ψw and the requirements for maintenance of phloem import.

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl2 to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by (31)P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na(+) and Mg(2+) are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K(+) is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg(2+) and Na(+), show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K(+), the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.

Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity

Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl(-)) and 87 millimolar (Na(+)), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K(+)/Na(+) and NO(3) (-)/Cl(-) uptake by roots.

Compartmentation of Solutes and Water in Developing Sugarcane Stalk Tissue

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from -0.6 to -1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from -1.0 to approximately -2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.

The effect of water stress on the vacuole‐extravacuole compartmentation of proline in potato cell suspension cultures

Solute compartmentation in cells is an important component of metabolic regulation. There is only little information on how stress treatment of cells effects this component. Therefore, the effect of water stress [10% (w/v) PEG 6000] on the vacuolar‐extravacuolar proline compartmentation was studied in a cell suspension culture of Svlanum tuberosum L, cv, HH258, In non‐stressed cells 34% of the total cellular proline was located in the vacuole. After 20 h of water stress the proline pool of the cells was increased 4‐6 fold and only t6% of it was found in the vacuole. A negative correlation between the total cellular proline content and its percentage in the vacuole was observed, irrespective of the culture method (stress or non‐stress culture). The stress‐induced changes in proline compartmentation are discussed.

Metabolic Changes Associated with Adaptation of Plant Cells to Water Stress

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from (15)N labeling kinetics after substituting [(15)N]ammonium and [(15)N]nitrate for the (14)N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and gamma-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and gamma-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and gamma-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low (15)N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.

Accumulation and subcellular distribution of cations in relation to the growth of potassium‐deficient barley

Abstract Growth of barley (Hordeum vulgare L., cv. Georgie) was insensitive to soil K content above about 150 mg kg−1, but at lower levels it declined. The reduction in yield was greater in soils containing approximately 10 mg Na kg−1 than in soils with about 90 mg kg−1 of Na. Growth was unaffected by changes in shoot K concentration above 75 mol m−3, but declined at lower concentrations, and the decrease was less in plants grown in soils with high Na. Growth responses were not simply related to tissue K concentrations because plants grown in soils with extra Na had higher yields but lower K concentrations.When soil Na was low, plants accumulated Ca as tissue K declined, but when Na was provided this ion was accumulated. Plant Mg concentrations were generally low but increased as K decreased. The Ca and Mg were osmotically active. There were highly significant inverse linear relationships between yield and either the Ca or Mg concentrations in the shoots.X‐ray microanalysis was used to examine the compartmentation of cations in leaves from barley plants (cv. Clipper) grown in nutrient solutions with high and low K concentrations. In plants grown with 2.5 mol m−3 K, this was the major cation in both the cytoplasm and vacuole of mesophyll cells. However, in plants grown with 0.02 mol m−3 K it declined to undetectable levels in the vacuole, although it was still detectable in the cytoplasm. In all plants, Ca was mainly located in epidermal cells. The implication of the results for explaining responses to K. in terms of compartmentation of solutes is discussed.

Comparison of Hevea Tonoplast Adenosine-triphosphatase from Freshly Isolated Vacuoles and Lyophilized Tonoplast Vesicles

Summary There is no significant difference between the ATPase present in the Hevea tonoplast of freshly isolated vacuoles and of lyophilized tonoplast vesicles (same k m value for MgATP 2- , same pH-dependence, same effect of inhibitors, same effect of ionophores and protonophores). It is suggested that the components of the protonmotive force regulate the activity of the tonoplast H + -translocating ATPase. The consequences of such effects may be very important in the regulation of events linked to the compartmentation of solutes inside the plant cell.

Solute distribution in Suaeda maritima

The distribution of sodium, potassium and glycinebetaine in shoot tissues of salt-treated Suaeda maritima was examined by semi-micro techniques after extraction into toluene-water. Much higher K/Na ratios were observed in the apical regions and in axillary buds than in more mature, fully vacuolated tissues. The younger tissues also contained very high levels of glycinebetaine. Electron-probe X-ray microanalysis of bulkfrozen and fractured preparations showed higher K/Na ratios and higher levels of sulphur and phosphorus in the cytoplasm of leaf primordial cells than in vacuoles of either young or old leaves, although the total counts were higher in the vacuolar samples. The results are discussed in relation to current models of subcellular solute compartmentation and salt tolerance in the Chenopodiaceae.

Osmotic adaptation in porphyra purpurea: use of 86Rb + and 42K + exchange kinetics to investigate intracellular solute compartmentation

The effects of salinity upon the intracellular compartmentation of K + have been studied using the radioisotopes 86Rb + and 42K +. Experimental evidence suggeststhat intracellular K + is mainly located within a noncytoplasmic cellular compartment (composed of a vacuolar and a chloroplast fraction). An overall reduction in the K + cotent of cytoplasmic and non-cytoplasmic compartments was observed in hyposaline media. However, the relative proportion of non-cytoplasmic K + was found to increase in dilute seawaters.

Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles

Vacuoles isolated from storage root tissue of red beet (Beta vulgaris L.) do not leak significant quantities of betanin, sucrose, Na(+) or K(+) during isolation. This indicates that analysis of vacuoles in vitro gives meanigful information about the compartmentation of solutes in vivo. Preparations of vacouoles were used to determine the distribution of glycinebetaine and proline between vacuole and cytoplasm in beet cells. Both compounds were detected in preparations of isolated beet vacuoles. In the case of glycinebetaine it was shown that this solute was associated with the vacuoles, not with the small number of other organelles which contaminated the preparations. The vacuolar pool accounted for 26 to 84% of the total tissue glycinebetaine and 17 to 57% of the proline. Concentrations of these compounds in vacuole and cytoplasm were calculated and were always higher in the cytoplasm than in the vacuole. The concentration gradient across the tonoplast varied considerably. The significance of these results is discussed in relation to the hypothesis that glycinebetaine and proline function as benign cytoplasmic osmotica.