Effect of fusicoccin on proton co-transport of sugars in the phloem loading of Ricinus communis L.

The loading of [¹⁴C] sucrose into the phloem from the apoplast of hollow Ricinus petioles was stimulated by fusicoccin (FC, 10 mg l⁻¹), by indole-3-acetic-acid (IAA; 10⁻² mol m⁻³) and inhibited by abscisic acid (ABA 10⁻² mol m⁻³) when added to a buffered perfusing solution of 2% sucrose and 30 mol m⁻³ KCl. A proton efflux was detected in the hollow petiole which was stimulated by FC in the presence of K⁺, insensitive to IAA, and inhibited by ABA, when present in the perfusing solution.The observed results are consistent with an H⁺/K⁺ exchange between the phloem sap and the apoplast which is responsible for the high pH and high [K⁺ of phloem saps. The resultant pH gradient between the phloem sap and the apoplast provides the energy for the proton co-transport of sucrose in phloem loading.     

Perfusion of onion root xylem vessels: a method and some evidence of control of the pH of the xylem sap

We describe a method for perfusing the xylem in the stele of excised onion roots with solutions of known composition under a pressure gradient. Tracer studies using [¹⁴C] polyethylene glycol 4000 and the fluorescent dye, Tinopal CBSX, indicated that perfusing solutions passed exclusively through the xylem vessels. The conductance of the xylem was small over the apical 100 mm of the root axis but increased markedly between 100 and 200 mm. Unbuffered perfusion solutions supplied in the range pH 3.7–7.8 emerged after passage through the xylem adjusted to pH 5.2–6.0, indicating the presence of mechanisms for absorbing or releasing protons. This adjustment continued over many hours with net proton fluxes apparently determined by the disparity between the pH of the perfusion solution and the usual xylem sap pH of about 5.5. Mild acidification of the xylem sap by buffered perfusion solutions increased the release of ⁸⁶Rb (K⁺) and ³⁵SO₄ ^2- from the stelar tissue into the xylem stream. The ion-transporting properties of onion roots seemed little changed by excision from the bulbs, or by removal of the apical zones of the root axis. The pH of sap produced by root pressure resembles that found in the outflow solutions of perfused root segments.     

Monitoring uptake and contents of Mg,Ca and K in Norway spruce as influenced by pH and Al,using microprobe analysis and stable isotope labelling

In a model system using intact spruce trees (Picea abies [L.] Karst.) we followed the path of magnesium, calcium and potassium during uptake into the root and during long-range transport into the shoot, by multiple stable isotope labelling. The roots of two- and three-year-old spruce trees originating from soil culture were removed from the soil and, in part or in toto, exposed to labelling solutions containing the stable isotopes ²⁵Mg or ²⁶Mg, ⁴¹K and ⁴²Ca or ⁴⁴Ca. Optical-emission-spectroscopy (ICP-OES) of plant fractions and labelling solutions was combined with the quantitative analysis of stable isotope ratios in sections of shock frozen, cryosubstituted material using the laser-microprobe-mass-analyser (LAMMA). This combination allowed us to distinguish, both in bulk samples and on the cellular level between (i) the fraction of elements originally present in the plant before the start of the labelling, (ii) the material taken up from the labelling solution into the plant and (iii) any material released by the plant into the labelling solution.In single-root labelling experiments, roots of three-year-old spruce trees, grown in nursery soil, were exposed to various pH conditions. The exchange of Mg and Ca with the labelling solution was nearly 100% in the cell walls of the mycorrhized finest roots. This exchange was only slightly affected by a step down to pH 3.5. The absolute Mg and Ca content in the cell walls was moderately reduced by incubation at pH 3.5 and strongly reduced in the presence of Al at this pH. After a pH 3.5 and 2 mM Al treatment we found Al in the xylem cell walls and the cortex cell lumina at elevated concentrations. To analyse the combined effect of high Al and high proton concentrations on the long-range transport, we used a split-root system. The root mass of an intact two-year-old spruce tree, grown in mineral soil, was divided into even parts and both halves incubated in solutions with two sets of different stable isotopes of Mg and Ca (side A: no Al, ²⁵Mg and ⁴²Ca; side B: +Al, ²⁶Mg and ⁴⁴Ca) and ⁴¹K on both sides. We observed a large uptake of Mg, Ca and K into the plant and a pronounced release. The net uptake of all three elements was lower from the Al-doted solution. In cross-sections of the apical shoot we found after seven-day labelling period about 60–70% of the Mg and Ca and 30% of the K content in the xylem cell walls originating from both labelling solutions. The clear majority of the Mg and Ca label originated from the Al-doted side.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

Glucosamine-mediated detoxification of p-benzoquinone and its removal by chitosan

p-Benzoquinone non-enzymatically reacted with d-glucosamine at physiological pH and moderate temperature. The reaction of p-benzoquinone with glucosamine was signaled by changes in the UV and visible spectra. The reactivity proceeded fastest at pH values above 7, with a sharp drop from pH 6.5 to 7.0, and the reaction was negligible in acidic conditions. The order of reactivity of amino sugars was d-mannosamine > d-glucosamine > d-galactosamine. From the reaction mixture, four conversion products were isolated and none was toxic to Escherichia coli even at 500–700 g ml^–1, while p-benzoquinone was cytotoxic to E. coli at 20 g ml^–1. Chitosan could react with p-benzoquinone efficiently and remove this toxicant in aqueous solution.     

Mechanism of sugar retention by roots of intact maize and field bean plants

The re-uptake of sugars driven by the proton gradient was studied in sugar net-release and net-uptake experiments using roots of intact maize (Zea mays cv. Blizzard) and field bean (Vicia faba L. cv. Alfred) plants. The net release of sugars into the root medium (0.1 mM CaSO₄) was stimulated by: the protonophore CCCP (10 M); the sulfhydryl reagent NEM (300 M); the specific inhibitor of plasmalemma ATPase vanadate (0.5 mM); and the inhibitor of the glucose carrier phlorizin (2 mM). Net uptake of glucose, fructose and arabinose from 10 M external concentrations was also inhibited by these substances. Surprisingly fusicoccin, a stimulator of net proton release did not effect net sugar uptake. Medium pH values only influenced sugar net uptake if the pH was above 7. It is concluded that a degradation of the proton gradient across the plasmalemma stimulates net sugar release because of disturbed re-uptake of sugars (in particular glucose) via a proton/sugar cotransport system. Thus, the retention of sugars by root cells not only depends on the plasmalemma permeability but also on the electro-chemical proton gradient. If an electro-chemical proton gradient is established by plasmalemma ATPase activity the re-uptake of sugars by proton/sugar cotransport minimizes the release of sugars into the rhizosphere.     

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with ¹⁴C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 M abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.Abbreviations ABA abscisic acid - FW fresh weight - IAA indole acetic acid - pd membrane potential difference - 1× perfusing solution (see methods) - H⁺ electrochemical proton gradient - pd solute-induced maximum depolarization of the membrane     

Changes in soluble carbohydrates during phytochrome-regulated petiole elongation in watermelon seedlings

Changes in soluble carbohydrate composition and concentration in leavesand petioles of watermelon (Citrullus lanatus (Thunb)Matsum and Nakai cv. Sugar Baby) seedlings during early stages ofphytochrome-regulated petiole elongation were investigated. Watermelon seedlingswere grown in a controlled environment with 350 molm^–2 s^–1 photosynthetically activeradiation (PAR) during a 12-h photoperiod. Low intensity end-of-day(EOD) light treatments (for 15 min) of red (R), far-red (FR) and FRfollowed by R (FR/R) were initiated when the seedlings were 14 days old.Seedling growth, and soluble carbohydrate concentration and composition inleaves and petioles were determined after 3 and 6 days of EOD light treatments.The EOD FR increased the petiole length and dry mass partitioned to petioles asearly as 3 days into the treatment. This increased petiole dry mass inFR-treated plants was accompanied with an increase in reducing sugar (glucoseand fructose) concentration in the petioles. Although both leaves and petiolesshowed this effect, the relative increase was greater in petioles than leaves.While the most abundant sugars in petioles were fructose and glucose, thepredominant sugars in leaves were sucrose, raffinose, and stachyose. Thephotoreversion of FR induced changes in growth and sugar concentrations by Rindicates the involvement of phytochrome in these processes.     

The contribution of tonoplast and plasma membrane to the electrical properties of a higher-plant cell

Translocation of ¹⁴C-labelled assimilates down the petioles was studied in intact plants of Pelargonium zonale (L.) L'Hérit ex Ait. The central bundle of the petiole was dissected out and treated with solutions of various inhibitors. Whereas cytochalasin B had no effect on ¹⁴C-translocation, a distinct and localized inhibition was caused by CCCP (10^-7 M), antimycin (5×10^-5 M), atractylate (5×10^-5 M), and valinomycin (10^-5 M) without any significant change in the proportion of [¹⁴C]sucrose in the translocate. The inhibition of translocation is inferred both by accumulation of ¹⁴C distal to and a decrease in ¹⁴C concentration basal to the treated petiolar region. If valinomycin was fed into the transpiration stream by flapping the peripheral bundles of the petioles an increased labeling of sugar phosphates occurred in the ¹⁴C fed leaf. Plasmolysis tests indicated that whereas CCCP interfered with the semipermeability of phloem cell membranes, valinomycin had no such effect. The results with valinomycin suggest a compartmentation of potassium ions for the translocation process but are ambiguous as to whether or not a potassium pump is involved.Dedicated to Wilhelm Halbsguth, Kiel to his 65th birthday     

Xylem perfusion of tap root segments of Plantago maritima: the physiological significance of electrogenic xylem pumps

Abstract A method is described for perfusing xylem vessels in tap root segments of the halophyte P. maritima. Use of excised segments allowed recording of the trans-root potential (TRP) at both ends of a segment. It was shown that there can be a spatial variation of electrogenic ion pump activity along the xylem in one root segment. The pH of perfusion solutions, differing in buffering capacity, was adjusted by the root segment to pH 5.1–5.6 during How through the xylem. This pH range was similar to that of sap produced by root pressure. The K⁺ activity in the outflow solution (K⁺_out) was rather constant at 12–13 mol m^?l3 despite input K⁺ activities ranging from 8 to 20 mol m^?l3. Addition of fusicoccin (10^?l2 mol m^?l3) to the perfusion solution induced a strong acidification of the xylem sap, a decrease in K⁺_out and an increase in Na⁺_out. Inhibition of aerobic respiration through anoxia inhibited electrogenic proton pumping into the xylem and led to an increase in K⁺_out and a decrease in Na⁺_out. It is suggested that transport of K⁺ and Na⁺ to the shoot of the halophyte P. maritima is regulated in the tap root by means of ion exchange between xylem vessels and xylem parenchyma and that this exchange is energized by proton translocating ATPases.     

Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H⁺ and K⁺ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.     

Bicarbonate transport inChara corallina: evidence for cotransport of HCO 3 − with H+

Summary Experiments were undertaken on the fresh water algaChara corallina to determine the form of inorganic carbon (CO₂ or HCO ₃ ^– ) which enters the cell during photosynthesis at alkaline pH. Recent proposals have centered on the possibility that proton efflux in alkaline solution is able to generate, in the immediate vicinity of the cell, a sufficiently low pH to raise the partial pressure of CO₂, and hence facilitate its passive permeation into the cell. Predictions have been made by modelling this situation (N.A. Walker, F.A. Smith & I.R. Cathers, 1980,J. Membrane Biol. 5751–58, J.M. Ferrier, 1980,Plant Physiol. 661198–1199), and these were tested by placing recessed-tip pH microelectrodes in the unstirred layer surrounding cells in stagnant solution (bulk pH 8.2, buffered only with 1mm HCO ₃ ^– ). Even as close as 2 m from the cell wall, the pH was typically 7.2 to 7.6 in the acid band center — over 1 pH unitgreater than that suggested by the models for CO₂ entry at the necessary rate for C-fixation. Further evidence for the entry of HCO ₃ ^– , rather than CO₂, at high solution pH was obtained from experiments in which the radial pH gradient in the unstirred layer was reduced. Buffer solutions containing 5mm phosphate or 5mm HEPES, raised the pH at the cell surface in the acid regions from around 7.2 to 7.8 or higher. This pH increase (reduction in acid gradient) would have greatly reduced the CO₂ level at the cell surface and should, therefore, have greatly reduced the CO₂-related¹⁴C-influx. However,¹⁴C-fixation was reduced by only 31% (phosphate) or 15% (HEPES), compared with buffer-free controls. Reduction of the unstirred layer thickness by fast solution flow resulted in a stimulation, and not a reduction, of¹⁴C-fixation. The similarity of our radial pH profiles near the wall with that predicted by the model (Walker et al., 1980) assuming H⁺–HCO ₃ ^– cotransport, together with the effects of buffer, and the results of increased solution flow rate, lead to the conclusion that cotransport of HCO ₃ ^– with H⁺ is the likely method of entry of inorganic carbon. Longitudinal pH profiles of theChara cell were obtained at a distance of 25 m from the wall. These revealed much sharper delineation of the acid and alkaline bands than has previously been possible with miniature pH electrodes. Profiles of local electric field, obtained with a vibrating probe, were in excellent agreement with the high resolution pH profiles. This supports the hypothesis that membrane proton transport has a role (direct) in the generation of the extracellular currents.     

Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K⁺-dependent, amiloride-sensitive K⁺/H⁺ exchange system.Abbreviation C _a element concentration related to water - C _d element content related to dry weight - dw dry weight - DMSO dimethylsulphoxide - emf electromotive force - NBD-Cl 7-chloro-4-nitrobenz-2-oxa-1,3-diazole - NEM N-ethylmaleimide - NMDG⁺ N-methyl-d-glucamine - PD potential difference - pH_i intracellular pH value - pH_lu luminal pH value - pmf protonmotive force - SD standard deviation - SE standard error - STEM scanning transmission electron microscopy - V _a apical potential difference - V _b basal potential difference - V _t transepithelial potential difference     

Effects of pH and heavy metal concentrations in solution culture on the proton release,growth and elemental composition ofAlyssum murale andRaphanus sativus L.

The proton release by a species that can hyperaccumulate nickel (Alyssum murale) and by a non-accumulator (Raphanus sativus L.) was studied at different pH and heavy metal concentrations in solution culture. Both factors influenced the growth and composition of the plants.A. murale was more sensitive than radish to a decrease of pH from 7.0 to 6.0 in the growth medium; plant yield and proton production diminished with decreasing pH. However, yields and proton production of radish only decreased at pH 5.5. The differences in the amounts of protons produced between the hyperaccumulator species and radish were not large enough to conclude that decreasing pH in the rhizosphere ofA. murale is a mechanism for heavy metal solubilization.Nickel concentrations inA. murale followed the typical pattern of an accumulator plant — more Ni was accumulated in the shoots than in the roots. Lower concentrations of Zn and Cd occurred in the shoots than in roots ofA. murale, and also of Ni in radish. The concentrations of Co inA. murale shoots were increased when Zn, Ni and Cd were absent from the nutrient solution. However, Co concentrations in radish shoots were independent of the concentrations of other heavy metals in the growth medium.     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

Proton-stimulated opening of stomata in relation to chloride uptake by guard cells

The concentration of potassium chloride required in the incubation medium to open stomata in isolated epidermal tissues of Commelina communis L. and Vicia faba L. could be lowered from 100 mM to 10 mM if the proton concentration of the ambient solution was increased from pH 5.6 to pH 3.5. This acidification effect was formerly attributed to the destruction of epidermal and subsidiary cells resulting in a relief of back pressure upon guard cells. While guard cells remain viable at pH 3.5, as demonstrated by their susceptibility to inhibition by uptake of glucose or to uncoupling by DNP, incipient destruction of the cells surrounding them could first be observed 30 min after the onset of the incubation experiment. By this time, however, the stomata had already opened; the time course of stomatal opening at pH 3.5 did not show any lag phase corresponding to the time required for damaging epidermal cells and showed no difference to that at pH 5.6 Thus, the acid-stimulated opening of stomata appears to be a biphasic phenomenon consisting of a physiologic effect onto which the physical effect of the relief of back pressure is superimposed over longer periods of incubation. To interpret the physiologic role of an increased proton concentration in the ambient solution of isolated epidermal strips, it is suggested that guard cells take up protons and chloride ions in an electroneutral symport. While protons are extruded again to generate the negative membrane potential required for potassium influx, chloride ions are retained to maintain electroneutrality.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

On the Relation Between the Effects of Auxin on Growth, pH and Potassium Transport

The relation between the effects of auxin on growth, pH and potassium transport in hypocotyl segments of Helianthus annuus was studied. In a solution containing 20 mM Na₂SO₄ auxin-induced growth was accompanied by an auxin-induced pH drop in the medium. (NH₄)₂SO₄, at the same concentration, brought about an almost complete abolishment of the effect of auxin on the pH. The magnitude of auxin-induced growth was, however, only slightly reduced. This result does not confirm the hypothesis according to which the action of auxin on growth is a result of its effect on the pH. In a solution containing 2 mM sodium phosphate buffer an inhibitory action of IAA on the release of potassium from the tissue was observed. Addition of 20 mM Na₂SO₄ to the medium brought about a complete abolishment of this effect. The magnitude of auxin induced pH drop was, however, similar in the two treatments. It was concluded that, although under suitable experimental conditions, a close relationship may exist between the effects of auxin on pH on K⁺ transport, the coupling between the two phenomena is not obligatory.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

A Protease Inhibitor from Penicillium cyclopium

A protease inhibitor produced by Penicillium cyclopium on solid cultures of wheat bran was purified by means of column chromatography on Duolite A-2 and DEAE-cellulose, acetone precipitation and lyophilization. The purified inhibitor obtained as a white, floccose and hygroscopic substance was monodisperse by ultracentrifugal analysis. It was found to be an acidic macro-molecule of a molecular weight of about 5000. The chemical analyses rejected the possibility of the presence of amino acids, peptides, sugars, amino sugars, or uronic acids in the inhibitor molecule.

Properties of a protease inhibitor from Penicillium cyclopium were studied. The pH range of the inhibitor action is restricted to acid pH, optimally at pH 3. Increasing temperature accelerates its action upon enzyme. The inhibitor causes enzyme inactivation in proportion to its concentration. It is fairly stable in an acid solution but unstable in an alkaline solution. It undergoes destruction by heat, hydrogen peroxide and ascorbic acid. The inhibitor reversibly combines with Al³⁺, Fe³⁺, Ag⁺ and Cu²⁺ to produce a precipitate. Salts interfer with the inhibitor activity. Generally, acid proteases from various penicillia are susceptible to the inhibitor while those from other genera are resistant.