Armen Kurkdjian

Regulation of Intracellular pH in Plant Cells

Publisher Summary This chapter reviews the basic principles of the techniques used for intracellular pH measurement in the main cell compartments—namely, cytoplasm and vacuole. Alongwith being both substrate and product in numerous metabolic reactions, protons fulfill the regulatory role of coordinating the activities of enzyme-catalyzed pathways, membrane transport, and other regulators. The protons connect cellular compartments and also play important roles in intercellular traffic. The sudden pH shifts may impose critical loads on the cells. The technical improvements that have been brought about since the previously published reviews are described in the chapter. The technique involves the extraction of cell sap and the measurement of its pH with a glass electrode. It is used for various types of plant materials. The principle of this technique is based on three requirements: the probe molecule is metabolically inert, only the uncharged form is membrane permeant, and the probe is not to change the pH of the respective compartment. The distribution of protons within a plant cell appears as a critical element of cell organization and function.

Characterization of an anion-permeable channel from sugar beet vacuoles: effect of inhibitors

The vacuole occupies 25‐95% of the plant cell volume and plays an essential role in maintaining cytoplasmic homeostasis of nutrients and ions. Recent patch‐clamp studies identified ion channels and electrogenic pumps as pathways for the movement of ions and metabolites across the vacuolar membrane (tonoplast). At high cytoplasmic Ca2+ (>10‐6 M) and negative potentials (inside the vacuole) non‐selective channels of the ‘slow‐vacuolar (SV)‐type’ were activated resulting in anion release or cation influx. In the present study these vacuolar channels were characterized pharmacologically by ion channel inhibitors. The cation‐transport inhibitors Ba2+, TEA+ and amiloride caused only partial and reversible block of the ‘SV‐type’channels, whereas anion‐transport inhibitors strongly affected the vacuolar channels. Pyridoxalphosphate and the dimethylaminecarboxylate derivates anthracene‐9‐carboxylic acid and C 144 reversibly blocked the channels up to 70% and Zncl2 up to 95%. DIDS and SITS inhibited this channel irreversibly up to 95%. The block developed under a variety of experimental conditions using solutions containing combinations of permanent cations and anions. The DIDS binding site is located on the cytoplasmic surface of the tonoplast, as intravacuolar DIDS did not block the channels. DIDS concentrations in the micromolar range, efficient in blocking 70—80% of the ‘SV‐type’ channels did not significantly affect ATP‐induced or pyrophosphate‐induced proton‐pumps. Stilbene derivatives may therefore be useful tools for studies on the substrate binding site on this vacuolar channel and for channel isolation.

Comparative studies on the electrical properties of the H+ translocating ATPase and pyrophosphatase of the vacuolar-lysosomal compartment

The electrical properties of the vacuolar‐lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole‐cell configuration of the patch‐clamp technique. Both pumps, the proton‐translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP(i)), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was about 2.5 microA/cm2 and about 0.5 microA/cm2 for the pyrophosphatase. K(m)ATP was 0.6 mM; K(m)PPi was 15‐20 microM with progressive inhibition above 150 microM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10,000‐fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO(3)‐ only.

Role of the Differentiation of Root Epidermal Cells in Nod Factor (from Rhizobium meliloti)-Induced Root-Hair Depolarization of Medicago sativa.

The stage of differentiation of epidermal cells and the development of root hairs was found to be important for the induction of depolarization in root hairs of Medicago sativa by Nod factor [NodRm-IV(S)] isolated from the bacterium Rhizobium meliloti. The electrical membrane response was concentration dependent, having its major effect (amplitude of the depolarization and number of root hairs that responded) at 10–8 and 10–7 M Nod factor. This response was correlated with a morphological effect of Nod factor in the root-hair-deformation bioassay at similar concentrations. The effect of Nod factor on depolarization and root-hair deformation showed specificity with respect to the structure, since unsulfated Nod molecules were inactive, as was the synthetic N,N′,N″,N‴- tetraacetylchitotetraose. The Nod factor that is O-acetylated at the nonreducing sugar was as efficient in root-hair deformation and membrane depolarization as the sulfated Nod factor.

Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles

Abstract The electrical properties of the tonoplast of mature sugarbeet root vacuoles have been studied using the patch-clamp technique. In whole-vacuole recordings, the addition of 5 mM Mg-ATP to the external solution activated a proton-translocating ATPase which produced inward currents of up to 65 pA. Furthermore, we identified a voltage-dependent membrane conductance which activated at hyperpolarized (inside-negative) potentials and decreased at positive potentials. Outside-out membrane patches predominantly contained a channel which showed an increasing probability of opening at potentials more negative than about –20 mV. These channels can account for the macroscopic currents recorded in whole vacuoles. The permeability sequence of the channel for cations and anions was: PKstaggered+ = PNa+ >PAc− >PNO3− >PMal2− >PCl−. The unit conductance of this channel was about 70 pS in symmetrical 50 mM KCl and 180 pS in symmetrical 200 mM KCl solutions. Another channel type of smaller conductance (15 pS in 50 mM KCl) was also present, but its properties have not yet been studied. The permeability sequence of the nonselective channel corresponds to that found by tracer measurements in vacuole suspensions, implying that the channel studied may present the molecular pathway for the movement of ions across the tonoplast.

A hydrogen ion-selective liquid-membrane microelectrode for measurement of the vacuolar pH of plant cells in suspension culture

Abstract H+-selective microelectrodes were made according to the method described by D. Ammann, F. Lanter, R. A. Steiner, P. Schulthess, Y. Shijo, and W. Simon (1981, Anal. Chem.53, 2267–2269). Some practical aspects of the preparation and use of these microelectrodes for in vitro and in vivo pH measurements in plant vacuoles have been examined. The trials indicate that the microelectrodes can be kept for up to 48 h without modification of their slope (mV/pH unit) and resistance. The H+-selective liquid can be used more than 4 months after being prepared. The vacuole is known to be the storage compartment of plant cells where solutes are accumulated; as an example, sucrose and a model protein, bovine serum albumin (BSA), were chosen to study the effect of solutes on the response of the microelectrodes. The results indicate that the slope of the regression line is not modified by sucrose (20–100 m m ) added to citrate buffer solution, but it is slightly decreased when the microelectrodes are tested in the presence of BSA or in plant juice, indicating that some components of the cell sap are able to interfere with and to modify the response of the microelectrodes. More experiments are needed in order to determini it proteins, ions, or another substance are the factors causing this effect. Microelectrodes with tip diameters in the range 0.3–0.6 μm and electrical resistance in the range 2 × 1012 ohms are shown to be suitable for the measurement of vacuolar pH in plant cells. Their short response time (several seconds) when inserted into vacuoles makes them appropriate for follwing vacuolar pH modifications in situ.

Intracellular pH in higher plant cells I. Improvements in the use of the 5,5-dimethyloxazolidine-2[14C], 4-dione distribution technique

Abstract The 5,5-dimethyloxazolidine-2,4-dione (DMO) distribution technique for the determination of intracellular pH has been improved to allow a more easy and accurate measurement of intracellular pH in Acer pseudoplatanus cells. These improvements concern the measurement of th amount of free intracellular DMO molecules when the diffusion equilibrium is reached and the measurement of the volume of the cells. The intracellular pH value calculated is an overall pH. The cytoplasmic and vacuolar pH have been estimated. The meaning and usefulness of this overall pH value is discussed.

Tryptamine uptake and accumulation by Catharanthus roseus cells cultivated in liquid medium

Abstract [ 14 C]tryptamine was rapidly accumulated by Catharanthus roseus cells cultivated in liquid medium. This lipophilic amine appears to enter the cells through a diffusion process, the cell membranes being much more permeable to the uncharged form of the weak base. As a consequence, tryptamine is probably mainly accumulated as a cation inside the large acidic vacuolar compartment and the accumulation is, at least in part, driven by the pH difference between the vacuole and the culture medium. Catharanthus roseus cells cultivated in liquid medium contained a large endogenous tryptamine pool which gave a high isotopic dilution of [ 14 C]tryptamine when this indole base was used as a precursor of alkaloid biosynthesis.

Evidence for an action of fusicoccin on the vacuolar pH of acer pseudoplatanus cells in suspension culture

Abstract The pH, malic acid and K + contents of the cell sap extracted from frozen thawed Acer pseudoplatanus cells were measured. A fusicoccin (FC) treatment of the cells induced an increase pf the pH and malic acid and K + contents indicating that a vacuolar pH modification was probably involved in FC action. Even though the decrease of the pH of the culture medium was measureable after a short period of FC treatment (10–15 min), at least 1 h was necessary for an increase of sap pH to be significant. The assumption of a vacuolar pH modification is supported by the modification of the distribution of [ 14 C] nicotine molecules between the cells and their culture medium induced by the FC treatment. As this lipophilic base is absorbed by diffusion of the neutral form and concentrated as ions in the acidic compartments of the cells (i.e. mainly the vacuole) FC action on nicotine accumulation can be interpreted as evidence for a vacuolar pH modification. Possible mechanisms of this FC action are tentatively discussed.

Evidence for an action of 2,4-dichlorophenoxyacetic acid on the vacuolar pH of Acer pseudoplatanus cells in suspension culture

Abstract Like plant organs, Acer pseudoplatanus cells grown in liquid medium responded to the action of auxin by acidifying their culture medium. As was the case for fusicoccin (FC) action, the decrease of extracellular pH was associated with an increase of the pH of the sap (pH s ) extracted from frozen-thawed cells and of the malic acid content of the cells. Furthermore, the distribution of [ 14 C]nicotine molecules between the cells and their culture medium was modified after 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. As this lipophilic base was absorbed by diffusion of the neutral form and concentrated as ions in the acidic compartment of cells (i.e. mainly the vacuole), 2,4-D action on nicotine accumulation could be interpreted as evidence for a vacuolar pH modification. Variations of the vacuolar pH-values during the growth of a cell population were shown with both methods of vacuolar pH measurement (freeze-thaw method or the method of absorption of [ 14 C]nicotine). The pH s -values depended on the initial concentration of auxin injected in the culture medium; pH s was always lower for cells grown on a low 2,4-D concentration as compared with cells grown on a high concentration of 2,4-D.