Concentrations of inorganic and organic solutes in extracts from individual epidermal,mesophyll and bundle-sheath cells of barley leaves

The distribution of solutes between epidermal, mesophyll and bundle-sheath cells in barley (Hordeum vulgare L. cv. Klaxon) leaves was studied by analysing extracts obtained from single cells with a modified pressure probe. Activity of the cytoplasmic marker enzyme, malate dehydrogenase, revealed that epidermal cell extracts were completely vacuolar in origin, but extracts from mesophyll cells also contained cytoplasmic constituents. The extracts were analysed for osmolality and the concentrations of K, Na, Ca, Cl, P, S, NO ₃ ^– , sugars and total amino acids. Epidermal and mesophyll cell extracts had similar osmolalities but these varied between 420 and 565 mosmol, kg ¹ depending on the leaf developmental stage; the osmolality of bundle-sheath extracts was approximately 100 mosmol, kg^–1 lower. Under the growth conditions used, K and NO ₃ ^– were found in all three cell types and their concentrations generally ranged between 180 and 230 mM. In contrast, Ca was almost restricted to epidermal cells, where it increased to 70 mM during leaf ageing. Phosphorus was only detectable ( 5 mM) in extracts from mesophyll and bundle-sheath cells, while Cl concentrations were highest in epidermal and lowest in mesophyll cell extracts. The concentrations of sugars and amino acids were close to the detection limit (approx. 2 mM) in epidermal cells but mesophyll cells contained total sugar (glucose, fructose and sucrose) of up to 78 mM and total amino-acid concentrations of up to 13.5 mM. Concentrations in bundle-sheath cells were intermediate between those in the epidermis and mesophyll.Abbreviations EDX analysis energy dispersive X-ray analysis - MDH malate dehydrogenase We wish to thank Paul Richardson, Jeremy Pritchard, Peter Hinde and Andrew Davies (Banger) for their helpfull discussion and technical advice. This work was financed by a grant (LR5/521) from the Agricultural and Food Research Council.     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Epidermal solute concentrations and osmolality in barley leaves studied at the single-cell level

The solute relations of the upper epidermis of the third leaf of barley (Hordeum vulgare L. cv. Klaxon) were studied by analysing vacuolar saps extracted from individual cells. Their osmolality (nanolitre osmometry) and the concentrations of K, Na, Ca, Cl, P, S (energy dispersive X-ray analysis) and NO ₃ ^– (microfluorometry) were measured. All of the osmotically important solutes were accounted for. These were K⁺, NO ₃ ^– , Cl^–, and Ca²⁺. The concentration of each solute varied along the leaf blade and changed with leaf age. Calcium in particular increased during leaf ageing, exceeding concentrations of 50 mM. Plants starved of Ca²⁺ during this period accumulated epidermal K⁺ instead of Ca²⁺. Leaf ageing was accompanied by an increase in epidermal osmolalities by about 100 mosmol · kg^–1. When compared to the bulk leaf extract, epidermal cell extracts exhibited significantly higher concentrations of NO ₃ ^– , Cl^– and Ca²⁺, similar concentrations of K⁺ and Na⁺, and lower concentrations of P. In plants subjected to various levels of NaCl stress (up to 200 mM), epidermal concentrations of Cl^– always exceeded those of the bulk extract, while Na⁺ concentrations were similar. Epidermal cells osmotically adjusted to the increase in the external salt concentration.Abbreviations EDX analysis energy dispersive X-ray analysis We wish to thank Paul Richardson, Jeremy Pritchard, Peter Hinde, Eirion Owen and Andrew Davies (Banger) for their helpfull discussion and technical advice. This work was financed by a grant (UR5/ 521) from the Agricultural and Food Research Council.     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Rapid and tissue-specific accumulation of solutes in the growth zone of barley leaves in response to salinity

The aim of the present study was to test whether rapid accumulation of solutes in response to salinity in leaf tissues of barley (Hordeum vulgare L.) contributes to recovery and maintenance of residual elongation growth. Addition of 100 mM NaCl to the root medium caused an immediate reduction close to zero in elongation velocity of the growing leaf 3. After 20–30 min, elongation velocity recovered suddenly, to 40–50% of the pre-stress level. Bulk osmolality increased first, after 60 min, significantly in the proximal half of the elongation zone. Over the following 3 days, osmolality increases became significant in the distal half of the elongation zone, the adjacent, enclosed non-elongation zone and finally in the emerged portion of the blade. The developmental gradient and time course in osmolality increase along the growing leaf was reflected in the pattern of solute (Cl, Na and K) accumulation in bulk tissue and epidermal cells. The partitioning of newly accumulated solutes between epidermis and bulk tissue changed with time. Even though solute accumulation does not contribute to the sudden and partial growth recovery 20–30 min after exposure to salt, it does facilitate residual growth from 1 h onwards. This is due to a high sink strength for solutes of the proximal part of the growth zone and its ability to accumulate solutes rapidly and at high rates.Abbreviations EDX analysis Energy-dispersive X-ray analysis - LEV Leaf elongation velocity - LVDT Linear variable differential transformer - REGR Relative elemental growth rate     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley leaves

The activities of two key enzymes of the glyoxylic-acid cycle, isocitrate lyase and malate synthase, can barely be detected in mature, presenescent primary leaves of barley (Hordeum vulgare L.) but are apparently induced in senescent leaf tissue. Upon incubation of leaf segments in permanent darkness, the activities appear and increase dramatically up to the sixth day and thereafter decline. The glyoxylic-acid cycle may thus be functional during foliar senescence. The main period of galactolipid loss is characterized by RQ values as low as 0.63, indicating that long-chain fatty acids produced from thylakoidal acyl-lipids may be utilized for gluconeogenesis involving corresponding glyoxisomal metabolic pathways. Foliar senescence may be characterized by a peroxisomeglyoxysome transition analogous to the glyoxisome-peroxisome transition in greening cotyledons of fat-storing seeds.Abbreviations FW fresh weight - MGDG monogalactosyl diacylglycerol - RQ respiratory quotient     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Protein turnover in the attached leaves of non-stressed and stressed barley seedlings

Protein turnover was examined, using tritiated water, in various 2-cm regions of 7-11-d-old, first leaves of barley (Hordeum vulgare). Differences were found between the regions in their protein turnover and their responses to stress. The rate constant for degradation for total protein was the same throughout the leaf and the average half-life (t_1/2) of protein=approx. 220 h. Only in the older regions did a 24-h pulse of³H₂O preferentially label protein with a t_1/2 (90 h) considerably shorter than the t_1/2 for total protein. Soluble protein was degraded faster than insoluble protein and contained an appreciable short-lived protein component observable by short-pulse labelling. The rate of protein synthesis was greatest in the cells of the youngest region and declined as each region aged. The mean rate of protein synthesis over the 4-d period was 4 and 7 nmol h^-1 of amino-N with respect to the regions 1–3 and 7–9 cm from the leaf tip. Seedlings, stressed by adding polyethylene glycol (2.0 MPa) to the roots, showed a marked loss of protein from the older leaf regions with only small losses in the younger regions. Amino acids accumulated in the younger region continuously whereas in the older region little accumulation occurred until day 3 of stress when proline levels increased. Protein synthesis was decreased by between 30% and 50% in all leaf regions. In the region 1–3 cm from the leaf tip, the rate of protein degradation of total protein was enhanced and equalled the rate of degradation of 24-h-pulse-labelled protein which was not itself significantly affected by stress (t_1/2=approx. 90 h). In the region 3–5 cm, the degradation of both 4-d and 24-h-labelled protein was enhanced by stress to rates similar to those found in the region 1–3 cm. This was largely through increases in the degradation of the insoluble protein, but the degradation of soluble protein was also raised. Protein degradation in the region 7–9 cm was not affected by stress.Abbreviations t_1/2 average half-life - PEG polyethylene glycol     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.     

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO₂. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO₂ assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO₂-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO₂-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO₂ assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. This work was supported by the Agricultural and Food Research Council, UK (Research grant PG50/67) and by the Science and Engineering Reserach Council, UK. C.A.L. was supported by the British Council, by an Overseas Research Student Award and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil.     

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [³H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [³H]metabolites and the other for autoradiography. The³H-activity remaining in the leaf tissue after washing out the water-soluble³H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [³H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [³H]PE and [³H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [¹⁴C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [¹⁴C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.Abbreviations and symbols PE phosphatidylethanolamine - PC phosphatidylcholine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - TLC thin-layer chromatography - _leaf leaf water potential     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves

Barley (Hordeum vulgare L.) plants were grown hydroponically with or without inorganic phosphate (P_i) in the medium. Leaves were analyzed for the intercellular and the intracellular distribution of P_i. Most of the leaf P_i was contained in mesophyll cells; P_i concentrations were low in the xylem sap, the apoplast and in the cells of the epidermis. The vacuolar concentration of P_i in mesophyll cells depended on P_i availability in the nutrient medium. After infiltrating the intercellular space of leaves with solutions containing P_i, P_i was taken up by the mesophyll at rates higher than 2.5 mol· (g fresh weight)^–1 · h^–1. Isolated mesophyll protoplasts did not possess a comparable capacity to take up P_i from the medium. Phosphate uptake by mesophyll protoplasts showed a biphasic dependence on P_i concentration. Uptake of P_i by P_i-deficient cells was faster than uptake by cells which had P_i stored in their vacuoles, although cytoplasmic P_i concentrations were comparable. Phosphate transport into isolated mesophyll vacuoles was dependent on their P_i content; it was stimulated by ATP. In contrast to the vacuolar P_i concentration, and despite different kinetic characteristics of the uptake systems for p_i of the plasmalemma and the tonoplast, the cytoplasmic p_i concentration was regulated in mesophyll cells within narrow limits under very different conditions of P_i availability in the nutrient medium, whereas vacuolar P_i concentrations varied within wide limits.Dedicated to Professor Wilhelm Simonis on the occasion of his 80th birthdayThis investigation was part of the research efforts of the Sonderforschungsbereich 176 of the Bayerische Julius-Maximilians-Universität Würzburg. We are grateful to Dr. Olaf Wolf for introducing us to the method for preparation of xylem sap of barley plants and to Mr. Yin Zuhua for fluorimetric experiments with the dye pyranine. T. Mimura is indebted to the Alexander-von-Humboldt-Stiftung for a postdoctoral research fellowship.     

Characterization of vacuolar polypeptides of barley mesophyll cells by two-dimensional gel electrophoresis and by their affinity to lectins

Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.Abbreviations IEF isoelectric focussing - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (-mannosidase, -galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the ¹⁴C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N-dicyclohexylcarbodiimide ([¹⁴C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF₀. Tonoplast polypeptides heavily labelled with [¹⁴C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - IA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate