Phosphate Uptake and Transport by Roots and Stolons of Intact White Clover (Trifolium repens L.)

The extent to which phosphate can be absorbed directly fromthe outer medium by stolon internodes and contribute to thetotal accumulation of phosphate by intact plants of white clover(Trifolium repens L. cv. Blanca) was assessed in hydroponicexperiments in a controlled environment room. The uptake ofphosphate by intact roots or stolons was measured by sealinga segment (6-0 mm long) across a flow-cell in which ³²P-labellednutrient solution was circulated for 24 h, the rest of the rootsystem receiving unlabelled nutrient solution. The rate of uptakeof phosphate (µmol g^–1 d^–1 dry wt. basis)by roots was more than 300 times that by intact stolons. Pretreatmentof stolons by gentle abrasion to remove cuticle, so as to simulatethe condition of stolons in the field, increased the uptakeof phosphate 7-fold compared with that of intact stolons. However,the potential of stolons to contribute to the P status of whitedover in the field was calculated to be small (5%). When an incision was made through the hypodermal layer of stolons,the rate of phosphate uptake greatly increased, attaining 71%of that by root segments. This increase, which was greater athigher phosphate concentrations, indicates that the suberi.zedhypodermis constitutes a major barrier to the influx of phosphatein the stolon. After withholding phosphate for different time intervals, thesubsequent rate of phosphate uptake by roots was increased 2-3-foldafter 2 d phosphate deprivation and 3-4-fold after 6 d or 13d phosphate deprivation. A higher proportion of absorbed phosphatewas transported to shoots in phosphate-deprived plants. After1 d of uptake following restoration of the phosphate supply,the concentrations of labelled phosphate in shoots were greaterthan in control plants, although the concentrations of labelin roots was less. However, the rate of uptake of phosphateby stolons, following deprivation, was not significantly increased.These results suggest that the mechanism regulating the enhancedrate of phosphate loading into the xylem, initiated by a periodof phosphate deprivation, is specific to roots and is not inducedin stolons. The results are discussed in relation to the growth and acquisitionof phosphate by white clover in the field. Key words: Nutrient deficiency, phosphate, stolons, transport (ions), Trifolium repens     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Direct Observation of Membrane Insertion by Enveloped Virus Matrix Proteins by Phosphate Displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes.     

Kinetics of Manganese Uptake by Intact Citrus Seedlings

Uptake of manganese by intact citrus seedlings can be represented by three phases of a single, multiphasic isotherm in the range 10^?8M–2× 10^?4M. The phases are separated by marked jumps and the kinetic constants increase upon transition to higher phases.     

Kinetics of Potassium and Magnesium Uptake by Intact Soybean Roots

The kinetics of uptake of K⁺ and Mg²⁺ were studied by using intact soybean [Glycine max (L.) Merr. cv. Amsoy] roots. Uptake of K⁺ in the concentration range 1.29 × 10^?5 to 1.82 × 10^?3 M can be represented by two phases of a single, multiphasic mechanism. Similarly, uptake of Mg²⁺ in the concentration range 4.10 × 10^?6 to 2.49 × 10^?4M was biphasic.     

pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts

Experiments in which [1-¹⁴C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C₃ plants.     

Nutrient Uptake by Different Parts of the Intact Roots of Plants

An apparatus is described for studying the uptake of ions byshort segments of intact root systems grown in water culture. When the entire root systems of young cereal plants are suppliedwith o'I ppm, P or Sr the quantities of both ions accumulatedin segments 3–5 mm long, or translocated from them toother tissues, are considerably smaller than those which movelongitudinally in the cortex for short distances. This process,which is under metabolic control, causes ions to be releasedto the external solution from parts of the root a few mm distantfrom the site of entry. The contribution, to the nutrition of barley plants 3–4weeks old, of different parts of the root system has been investigated.Between seminal axes, nodal axes, and laterals total uptakeper unit length of root varies largely, though not entirely,with volume. The ratio in which phosphate and strontium areabsorbed is not constant throughout the root system, the absorptionof phosphate being relatively greater by laterals. Little translocationoccurs from the apical 3 mm of roots and the fraction of theabsorbed ions translocated to shoots from older root segmentsis considerably greater for nodal axes than for seminal axesor laterals. The significance of the distribution of absorbing power throughoutthe root system is considered in relation to the nutrition ofplants grown in soil, especially when the rate of diffusionto the root surface may limit nutrient uptake.     

Effect of Ferricyanide on Potassium Uptake by Intact Epidermal Tissue and Guard Cell Protoplasts

The effect of ferricyanide on K^$ fluxes in epidermis and inguard cells of Commelina communis L. were studied. Ferricyanideenhanced guard cell protoplasts swelling, which results fromenhanced K^$ uptake. In intact epidermis ferricyanide inhibitedK^$ uptake and consequently, stomatal opening. This was foundin floated and submerged epidermal tissues, indicating thatthe degree of contact with the solution does not affect theresponse to ferricyanide. Investigation of the rate of plasmolysisand de-plasmolysis of guard cells in epidermal tissue revealedthat ferricyanide enhances deplasmolysis, caused by K^$ uptake,only in completely plasmolysed cells, which resemble protoplastsin situ. (Received January 21, 1988; Accepted March 24, 1988)     

Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of Escherichia coli culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.Of worldwide concern is the increasing development of bacterial and fungal strains that are resistant to currently available antimicrobial drugs. This worsening situation has spurred Herculean efforts to develop new classes of antibiotics with novel targets and modes of action (19). Cationic antimicrobial peptides (AMPs) play a key role in the primary host defense of living organisms against infections by pathogenic microorganisms. Because their mechanisms of antimicrobial action differ from those of conventional antibiotics, AMPs have received increasing attention as a potential new class of therapeutic substances (22, 30).In contrast to bacterial growth in the presence of commonly prescribed antibiotics, the growth of bacteria in the presence of AMPs does not easily give rise to the selection of pathogenic drug-resistant mutant strains. This is because AMPs rapidly kill microbes by a variety of mechanisms, including (i) fatal depolarization of the normally energized bacterial membrane, (ii) creation of physical holes that cause cellular contents to leak out, (iii) degradation of the cell wall, (iv) disturbance of membrane functions, and/or (v) damaging of critical intracellular targets after internalization of the AMPs (7, 11, 19, 22, 30). Moreover, AMPs activate the host''s innate (nonspecific) immune response without acting as a foreign antigen target of the host''s adaptive immune system (23, 30). Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means of mass production has limited the development of these peptides as human therapeutics (8).Numerous biological expression systems have been introduced for the cost-effective production of AMPs in Escherichia coli (9). To decrease their natural destructive behavior toward microorganisms and sensitivity to proteolytic degradation, AMPs are often produced as fusion proteins in heterologous hosts (12, 16). These studies show that certain fusion partner proteins neutralize the toxicity of AMPs and improve their stability against proteolysis in an expression host. In another series of experiments, recombinant AMP-containing fusion proteins are expressed in tandem repeats in an attempt to increase AMP production. As expected, multimeric expression further enhanced the yield of AMP fusion proteins (9, 12, 16). However, all of these methods require that the AMP be separated from its fusion partner, and recombinant fusion proteins, including multimeric ones, are usually cleaved with enzymes such as furin or chemicals such as CNBr (12, 16). This additional process results in inefficient cleavage and thus poor recovery of AMPs from fusion partners. Moreover, unwanted amino acid residue(s) are often included in the AMPs after the cleavage reaction and can decrease antimicrobial activity and cause problematic side effects (18). Therefore, a new approach for producing an intact and biologically active AMP without the inclusion of an enzymatic or chemical cleavage step is needed.We have developed here a novel translationally coupled, two-cistron expression system for the production of recombinant AMPs in their natural forms. Using this system, we were able to produce, from 1 liter of E. coli culture, ∼100 mg of a potent AMP, buforin IIb (BIIb) (15), without a cleavage step, and other cationic AMPs (parasin I [24] and pexiganan [6]) were also successfully produced.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Assessment of Noradrenaline and 5-Hydroxytryptamine Uptake in Intact Rat Brain

A method for the measurement of 3H-monoamine [noradrenaline (NA) and 5-hydroxytryptamine (5-HT)] uptake into intact brain following intracerebroventricular injection is described. Most of the accumulated monoamine was associated with nerve terminals (synaptosomes). Radioactivity in the synaptosomal fraction was retained by 0.45-micron filters and was osmotically sensitive, features indicative of accumulation within particles rather than binding to membranes. [3H]5-HT associated with synaptosomes was reduced in animals pretreated with the neurotoxin 5,7-dihydroxytryptamine, in parallel with a reduction in endogenous 5-HT levels. Oral administration of tricyclic antidepressants including clomipramine, desipramine, and imipramine inhibited the synaptosomal accumulation of 3H-monoamines. Overall, the features of the accumulation of 3H-monoamines, after intracerebroventricular administration, displayed many of the characteristics expected of a physiological monoamine reuptake system.     

Uptake of Inorganic Phosphate by Suspension-Cultured Tobacco Cells: Kinetics and Regulation by Pi Starvation

The kinetics of P_i uptake by phosphate-starved and non-starvedtobacco cells (Nicotiana tabacum BY-2) suspension culture wasinvestigated. The kinetic parameters of P_i uptake were determinedby computer simulation of the curve that represented the time-dependentloss of P_i from the culture medium. The uptake profile couldbe completely explained by assuming the existence of only onekind of Michaelis-Menten-type P_i-transport system with an affinityfor P_i (K_m) of about 2.5 µM (the lowest value reportedto date) in both P_i-starved and non-starved cells. No evidencewas obtained suggesting the existence of a "low-affinity" P_i-uptakesystem that has been postulated to exist in several other plantmaterials. The V_max for uptake of P_i by non-starved cells was12 nmol per minute per milliliter of packed cell. Phosphatestarvation increased the V_max more than 5-fold, while it hadno effect on the affinity for P_i. V_max began to increase (atan almost constant rate) just after loss of all P_i from theculture medium and it reached a maximum about 16 hours later.This induction process was completely prevented by the additionof cycloheximide to the culture medium. All these results suggestthat P_i starvation increases the synthesis of a phosphate-carriercomplex that is postulated to be involved in the P_i-uptake process. (Received August 12, 1994; Accepted December 26, 1994)     

Effects of Ionic Strength of Nutrient Solutions on Phosphate Uptake by Sunflower

Active phosphate uptake by the roots of young sunflower plants was stimulated nonspecifically by increasing the total salt concentration of the uptake solution. Inhibition of active uptake by DNP-treatment removed the salt stimulation. Independently of the rate of active uptake the amount of phosphate present in the free space of the roots increased as the salt concentration was raised. It is suggested that at low ionic strength of the nutrient solution the initial passive step of ion transport through the root free space can limit the overall uptake rate.     

Uptake of Gibberellic Acid in Intact and Scarified Seeds of Barbarea vulgaris

Uptake of gibberellic acid as a function of duration of exposure, external concentration, and seed lot was measured in seeds of yellow rocket (Barbarea vulgaris R. Br.) by means of lettuce hypocotyl bioassays of the acidic, basic, or neutral fractions of seed extracts and by uptake of ¹⁴C-GA₃. In intact seeds, where mM levels of GA₃ promoted only 25% germination, uptake was completed within 24 h of exposure. The maximum uptake was about 0.2% of external amount. Although germination promotion by GA₃ differed among seedlots of yellow rocket, relative uptake (percentage of the external GA₃) was nearly the same. The relative rate of uptake of GA₃ was similar for scarified and intact seeds, but germination was promoted in scarified seeds by much lower levels of GA₃ than in intact seeds. Total uptake in scarified seeds was much higher, however (about 10% of the external amount). In seeds imbibed in H₂O, practically no endogenous GA-like activity was detected in either the acidic, basic, or neutral fractions. It was also apparent that intact seeds could take up quantities of GA₃ that failed to promote germination, but were comparable to quantities that promoted germination in scarified seeds.     

局部供磷对玉米根系生长、磷吸收速率的影响

玉米幼苗种子根局部供磷可明显改变根系的形态。供磷区侧根生长增加,无磷区侧极生长减少。供磷区1次、2次侧根长度与2次侧根数量明显增加;而1次侧根数量则不增加。供磷区缩小时,根系生长加快,单位根区磷吸收速率增加,但单位根重磷吸收速率的增加不很明显。磷局部供应植株主要通过供磷区根系的生长来增加磷的吸收,以满足植株对磷的需求。局部供磷植株中转运到供磷根区的光合产物明显多于无磷根区。     

The Relation between the Synthesis of Inorganic Polyphosphate and Phosphate Uptake by Chlorella vulgaris

During a period of phosphate starvation, the phosphate contentof cells of Chlorella vulgaris which had been grown in phosphate-richsolution, decreased. The levels of most phosphate fractionsdeclined, especially those of inorganic polyphosphates, whichat first accounted for about 5 per cent of the total phosphateand virtually disappeared after 36 h starvation. On return toa phosphate medium, phosphate was taken up at a much fasterrate than before starvation, with a striking increase in acid-solublepolyphosphate. The stimulated phosphate uptake and polyphosphateincrease have been shown to be specific effects of phosphatestarvation, occurred only when excess phosphate was suppliedand required light or air for the provision of energy. Therewas relatively little change in the concentrations of otherphosphate fractions, including orthophosphate. Inorganic polyphosphatewas found to be synthesized solely from phosphate absorbed fromthe medium. It is argued that polyphosphate synthesis is a consequenceof the stimulation of phosphate uptake, induced by the starvationperiod.     

The Effect of Low Osmotic Potential on Phosphate Uptake and Metabolism by Beetroot Discs

The uptake of phosphate by aged beetroot discs was examinedunder a range of water deficits which were induced osmoticallywith mannitol or polyethylene glycol At low water potentialsthe absorption of labelled phosphate was enhanced when the ambientconcentration of phosphate was high (10^–3 M) and the timefor absorption was short (30 mm): this occurred even when themetabolism was inhibited (by the water deficit), as indicatedby lowered oxygen consumption and incorporation of ³³P intohexose-phosphate, phosphoglycenc acid, a liquid fraction, anda precipitate consisting mainly of cell walls. The Q₁₀ of theuptake process approached unity at these low water potentials.Accompanying the enhanced uptake there was an increase in leakageof previously absorbed phosphate. It is postulated that thestimulation in uptake of phosphate in low osmotic potentialswas due to an increase in permeability Over long periods (6 h) uptake was inhibited by treatment atlow osmotic potentials. This is interpreted in terms of inhibitionof a metabolic component of phosphate uptake.