Regulation of a voltage-dependent,calcium-activated K conductance by cyclic GMP in dissociated flounder enterocytes

Enterocytes from the winter flounder (Pseudopleuronectes americanus) were isolated by collagenase digestion and maintained in flounder Ringer's solution. Whole cell currents were studied using the amphotericin-perforated whole-cell patch clamp technique. The mean resting membrane potential and capacitance values or dissociated cells were-45±7 mV and 5±0.4 pF, respectively. Enterocytes held at-20 mV and treated with 1 mol·l^-1 ionomycin exhibited outward currents when cells were stepped through a series of voltages from-60 to +110 mV. The reversal potential of this current in flounder Ringer's solution was-55 mV and the voltage at which half-maximal activation occurred was +20 mV. Voltage-dependent inhibition of outward current was observed at +60 mV and above. When cells were bathed in symmetric K Ringer's solution the reversal potential shifted to zero mV and no inhibition of current was observed at voltages between-60 and 140 mV. When the holding potential of the cell was changed from-20 to-80 mV and stepped from-60 to +110 mV, a second [previously characterized, O'Grady et al. (1991)] K current with delayed-rectifier properties was identified. This observation demonstrated that the delayed rectifier K channel and the Ca²⁺-activated K channel described in this study exist in the same cell. Extracellular addition of 2 mmol·l^-1 Ba²⁺ to cells bathed in symmetric K Ringer's solution resulted in nearly complete inhibition of outward current. Charybdotoxin produced only minor effects on this current. Addition of 8-Br cGMP to the bathing solution also inhibited outward current and this effect could be partially reversed following washout of 8-Br cGMP from the bathing solution. The results of this study indicated that a Ca²⁺-activated K conductance in winter flounder enterocytes is potentially inhibited by agents that increase intracellular cGMP. A similar effect of cGMP on a delayed rectifier K channel in flounder enterocytes was previously demonstrated.Abbreviations ANP atrial natriuretic peptide - CTX charybdotoxin - EPPS N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid     

Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L.

Photoassimilating mature leaves of Cucumis melo exported carbon at a rate of 1.7 mg C·dm^-2·h^-1. Radiolabeling with ¹⁴C showed that stachyose and raffinose are the main carbohydrates translocated. Autoradiograms indicated that sieve elements of the abaxial phloem of minor veins are the sole conduits for carbon export from mature leaves and carbon import into immature leaflets. Sieve elements of the abaxial phloem are associated with intermediary cells which are intimately connected with the surrounding mesophyll cells by numerous plasmodesmata. Photoassimilate, labeled with ¹⁴C, was released into the leaf apoplast and could be trapped in a buffer solution circulating over the abraded adaxial epidermis. Carbon efflux was 1% of the carbon-export rate. A comparable distribution of ¹⁴C among the sugars, amino acids and organic acids, recovered from the free space and from leaf extracts, was recorded. The composition of released ¹⁴C-labeled carbohydrates in the free space resembled the pattern of photoassimilate, but differed clearly from the translocate. Release of organic compounds into the leaf apoplast was stimulated by chelating agents like Na-ATP, ethylenediaminetetraacetic acid and ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid; a correlation between carbon efflux into the apoplast and carbon export from the leaf was not detected. It is suggested that the release of organic compounds into the leaf apoplast of Cucumis melo is the consequence of a general leakage from mesophyll and vascular parenchyma cells. A selective release of transport oligosaccharides was not observed. The experimental results presented here do not preclude a symplastic transfer of assimilates in mature leaves.Abbreviations EDTA ethylenediaminetetraacetate - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PCMBS p-chloromercuribenzenesulfonic acid     

Turgor-dependent efflux of assimilates from coats of developing seed of Phaseolus vulgaris L.: water relations of the cells involved in efflux

The turgor-homeostat model of assimilate efflux from coats of developing seed of Phaseolus vulgaris L. was further characterised. The turgor pressure (P), the volumetric elastic modulus () and hydraulic conductivity (L_p) of the seed coat cells responsible for assimilate efflux and cotyledon storage parenchyma cells were determined with a pressure probe. In addition, turgor of the seed coat and cotyledons was estimated by measuring the osmolalities of symplastic and apoplastic fluids extracted by centrifugation. Osmolality of symplastic and apoplastic saps collected from the seed coat declined significantly over the period of seed development from a cotyledon water content of 80% to 50%. However, the difference in osmolalities of the apoplastic and symplastic saps remained relatively constant. For cotyledons, osmolality of the apoplastic sap exhibited a significant decline during seed development, while the osmolality of symplastic sap did not change significantly. Hence cotyledon P increased as the water content dropped from 80% to 50%. For both detached and attached empty seed coats, a small decrease (ca. 40mOsmol·kg^–1) in the osmolality of the bathing solution, led to a rapid increase in P of cells involved in assimilate efflux (efflux cells) by about 0.07 MPa. Thereafter, cell P exhibited a rapid decline to the original value within some 20–30 min. When P of the efflux cells was reduced by increasing the osmolality of the bathing solution, P exhibited a comparable rate of recovery for attached empty seed coats but there was no P recovery to its original value in the case of detached seed coats. In contrast, the cotyledon storage parenchyma cells did not exhibit P regulation when the osmolality of the bathing solution was changed. The observations that the efflux cells of P. vulgaris seed coats can rapidly adjust their P homeostatically in response to small changes in apoplastic osmolality are consistent with the operation of a turgor-homeostat mechanism. The volumetric elastic modulus () of the seed coat efflux cells exhibited a mean value of 7.3±0.8 MPa at P=0.15 MPa and was found to be linearly dependent on cell P. The e of the cotyledon storage parenchyma cells was estimated to be 6.1±1.0 MPa at P=0.41 MPa. Hydraulic conductivity (L_p) of the seed coat cells and the cotyledon cells was (8.2±1.5) × 10^–8m·s^–1·MPa^–1and (12.8±1.0) × 10^–8 m·s^–1·MPa^–1, respectively. The relatively high , i.e., low elasticity, for the seed coat cell walls would ensure that small changes in water potential of the seed apoplast will be reflected in large changes in cell P. The high L_p values for both the seed coat and the cotyledon cells is consistent with the rapid changes in P in response to changes in water potential of the seed apoplast.Abbreviations LYCH Lucifer Yellow CH - volumetric elastic modulus - L_p hydraulic conductivity - P turgor pressure - osmotic pressure - t_1/2 half-time for water exchange The investigation was supported by funds from the Australian Research Council. We are grateful to Louise Hetherington for competent technical assistance and to Kevin Stokes for raising the plant material.     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S _i) and the swelling coefficient (K ^cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pH_i = f (S _i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S _t ^cat) and the anion exchange (S _t ^an) groups were determined in the root cell walls. The number of the functional group of each type (S ^j) was estimated, and the corresponding values of pK _a ^j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK _a ^j = f (C ^K+)] were determined. It was found that the swelling of root cell walls changes with pH, C ^K+ and strongly depends on plant species. Within the experimental pH and C ^K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (S ^j, pK _a ^j, K ^cw), the equation {pK _a ^j = f (C^K+)} and the dependencies {K ^cw = f (C^K+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10^-8 to 5.5·10^-8 m·s^-1·MPa^-1 in wheat and from 0.9·10^-8 to 4.8·10^-8 m·s^-1·MPa^-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10^-7 m·s^-1·MPa^-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10^-7 m·s^-1·MPa^-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.Symbols L _{p, cell} cell hydraulic conductivity - L _{p, root} root hydraulic conductivity - L _{p, root} calculated root hydraulic conductivity - root reflection coefficient     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.     

Light-promoted changes in apoplastic K+ activity in the Samanea saman pulvinus,monitored with liquid membrane microelectrodes

The movement of Samanea saman (Jacq.) Merrill leaflets is a consequence of the re-distribution of K⁺ and anions between motor cells on opposite sides of the pulvinus. We used a K⁺-sensitive microelectrode to study dynamic changes in K⁺ transport through motor-cell membranes during and immediately after change in illumination. Potassium-ion-sensitive and reference microelectrodes were inserted into extensor or flexor tissue of a whole pulvinus in white light (WL). A brief pulse of red light (RL) followed by darkness (D) (a) increased K⁺ activity in the extensor apoplast, indicating K⁺ release by the protoplast; and (b) decreased K⁺ activity in the flexor apoplast, indicating K⁺ uptake by the protoplast. White light after 35–40 min D reversed K⁺ activity in the extensor apoplast to approximately its original value. Blue light substituted partially for WL in this regard. Potassium-ion activity in the flexor apoplast reverted to approximately its original value after 2 h, with or without white illumination. Our data support the hypothesis that K⁺ efflux from extensor cells and K⁺ uptake by flexor cells following a WLRLD transition occurs by way of K⁺ channels.Abbreviations L light - WL white light - RL red light - BL blue light - D darkness     

Effects of abscisic acid,gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.Abbreviations ABA abscisic acid - FC fusicoccin - GA₃ gibberellic acid - PD electric potential difference (between the vacuole and the external medium) - CH cycloheximide - DNP dinitrophenol - FCCP (p-trifluormethoxy)-carbonylcyanide-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide     

Agrobacterium rhizogenes-mediated transformation of Rubia peregrina L.: in vitro accumulation of anthraquinones

An Agrobacterium rhizogenes-mediated transformation system for Rubia peregrina L. has been established by co-cultivation of callus cultures or by direct infection of explants with A. rhizogenes LBA 9402 harbouring the binary vector pMON 9703 containing gus and npt-II genes as markers. The putative transformed roots were selected on medium containing kanamycin (25 mg l^-1). Antibiotic resistant root clones were subjected to histochemical analysis for the localisation of -glucuronidase activity. Polymerase chain reaction was used to confirm the presence of gus, npt-II and T _L border sequences in the transformed root clones. Spontaneous regeneration of shoots was observed from 30 day-old transgenic roots. Total anthraquinone and alizarin contents of transgenic root cultures were measured by spectrophotometry and by high performance liquid chromatography. The accumulation of total anthraquinones in transformed roots was found to be approximately 2-fold higher than that found in one year-old field grown roots (2.12±0.12 and 1.23±0.12 mg g^-1 dry weight, respectively). Alizarin was found to be the major anthraquinone in transformed root cultures and was found to be approximately 3-fold higher than in field grown roots.Abbreviations BA 6-benzyladenine - B5 Gamborg B5 medium - gus -glucuronidase gene - GUS -glucuronidase - HPLC high performance liquid chromatography - MS Murashige and Skoog medium - NAA -naphthalene acetic acid - npt-II neomycin phosphotransferase II gene - OD₆₀₀ optical density at 600 nm - PCR polymerase chain reaction - T _L left border sequence of T-DNA - vir D1 virulence D1 gene - YMB yeast mannitol broth     

Extent of intracellular pH changes during H+ extrusion by maize root-tip cells

³¹P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H⁺ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H⁺. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H⁺ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h^-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H⁺ extrusion with enhanced organic-acid synthesis or other intracellular responses to H⁺ pumping.Abbreviations FC fusicoccin - P_i inorganic phosphate - NMR nuclear magnetic resonance - chemical shift - MDP methylene diphosphonic acid     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

Effect of changes in water content on photosynthesis,transpiration and discrimination against 13CO2 and C18O16O in Pleurozium and Sphagnum

Photosynthetic gas exchange characteristics of two common boreal forest mosses, Sphagnum (section acutifolia) and Pleurozium schreberi, were measured continuously during the time required for the moss to dry out from full hydration. Similar patterns of change in CO₂ assimilation with variation in water content occurred for both species. The maximum rates of CO₂ assimilation for Sphagnum (approx. 7 mol m^–2 s^–1) occurred at a water content of approximately 7 (fresh weight/dry weight) while for Pleurozium the maximum rate (approx. 2 mol m^–2 s^–1) occurred at a water content of approximately 6 (fresh weight/dry weight). Above and below these water contents CO₂ assimilation declined. In both species total conductance to water vapour (expressed as a percentage of the maximum rates) remained nearly constant at a water content above 9 (fresh weight/dry weight), but below this level declined in a strong linear manner. Short-term, on-line ¹³CO₂ and C¹⁸O¹⁶O discrimination varied substantially with changes in moss water content and associated changes in the ratio of chloroplast CO₂ to ambient CO₂ partial pressure. At full hydration (maximum water content) both Sphagnum and Pleurozium had similar values of ¹³CO₂ discrimination (approx. 15). Discrimination against ¹³CO₂ increased continuously with reductions in water content to a maximum of 27 in Sphagnum and 22 in Pleurozium. In a similar manner C¹⁸C¹⁶O discrimination increased from approximately 30 at full hydration in both species to a maximum of 150 in Sphagnum and 90 in Pleurozium, at low water content. The observed changes in C¹⁸O¹⁶O were strongly correlated to predictions of a mechanistic model of discrimination processes. Field measurements of moss water content suggested that photosynthetic gas exchange by moss in the understory of a black spruce forest was regularly limited by low water content.     

A model for water transport in the stele of plant roots

Summary The mechanism of water movement across roots is, as yet, not well understood. Some workable black box theories have already been proposed. They, however, assumed unrealistic cell membranes with low values of , or were based on a poor anatomical knowledge of roots. The role of root stele in solute and water transport seems to be especially uncertain. An attempted explanation of the nature of root exudation and root pressure by applying the apoplast canal theory (Katou andFurumoto 1986 a, b) to transport in the root stele is given. The canal equations are solved for boundary conditions based on anatomical and physiological knowledge of the root stele. It is found that the symplast cell membrane, cell wall and net solute transport into the wall apoplast are the essential constituents of the canal system. Numerical analysis shows that the canal system enables the coupled transport of solutes and water into a xylem vessel, and the development of root pressure beyond the level predicted by the osmotic potential difference between the ambient medium and the exudate. Observations on root exudation and root pressure previously reported seem to be explained quite well. It is concluded that the movement of water in the root stele although apparently active is essentially osmotic.Abbreviations J _v ^ex volume exudation per root surface - J₀ non-osmotic exudation - L^r overall radial hydraulic conductivity of an excised root - reflection coefficient - C_s difference in the osmotic concentration between the bathing medium and the exudate - R gas constant - T absolute temperature - C_K molar concentration of K⁺ - C_Cl molar concentration of Cl^– - C_j molar concentration of ion species j - P_j membrane permeability of ion j - z_j valence of ion j - F Faraday constant - V_ix intracellular electric potential with reference to the canal     

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml^–1. A maximum production of 4.6 g recombinant tumstatin (10⁷ cells)^–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO₄.     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Non-mycorrhizal uptake of amino acids by roots of the alpine sedge Kobresia myosuroides: implications for the alpine nitrogen cycle

Non-mycorrhizal plants of the alpine sedge, Kobresia myosuroides, take up the amino acid glycine from nutrient solutions at greater rates than NO _inf3 ^sup- or NH _inf4 ^sup+ . The amino acids glutamate and proline were also taken up at high rates. Total plant biomass was twice as high after 4 months of growth on glycine, compared to NH₄NO₃, with significant increases in both root and leaf biomass. By taking advantage of differences in the ¹³C signature of air in the growth chamber and the glycine used for growth, a two-member mixing model was used to estimate that a significant amount of the glycine was taken up as intact molecules, enough to contribute 16% of the total carbon assimilation over a 4-month growing period. Glycine uptake was inhibited when roots were exposed to N₂ in place of air, and when the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to the root solution. From these results it is concluded that glycine uptake occurs through active transport. Glycine uptake exhibited a Q₁₀ of 2.0 over the temperature range 5–15° C, with relatively high rates maintained at the lowest temperature measured (5° C). Roots of Kobreasia were not capable of taking up NH _inf4 ^sup+ at measureable rates. To our knowledge, this is the first report of a plant whose non-mycorrhizal roots cannot take up NH _inf4 ^sup+ . Measurements of three N fractions (NO _inf3 ^sup- , NH _inf4 ^sup+ , and total amino acids) in the soil pore water were made over two growing seasons in two Kobresia dry meadows using microlysimeters. At the West Knoll site, which is characterized by soils with average amounts of organic matter, the dominant forms of N in the soil pore water were NO _inf3 ^sup- and NH _inf4 ^sup+ (0–450 mol L^-1). Amino acid concentrations were generally less than 20 mol L^-1 at this site. At the East Knoll site, which is characterized by soils with higher than average amounts of organic matter, amino acids were generally present at higher concentrations (17–100 mol L^-1), compared to NO _inf3 ^sup- and NH _inf4 ^sup+ . The most abundant amino acids were glycine (10–100 mol L^-1), glutamate (5–70 mol L^-1), and late in the season cystein (5–15 mol L^-1). The results demonstrate that this sedge, which dominates dry meadow communities in many alpine ecosystems, is capable of taking up intact amino acids as a principal N source, and has access to high amino acid concentrations in certain alpine soils. Such uptake of organic N may accommodate plant N demands in the face of slow alpine N mineralization rates due to cold soil temperatures.