Stimulation of H+ efflux and cation uptake by fusicoccin in barley roots

193

Proton extrusion by roots of intact sunflower plants (Helianthus annuas L.) was studied in nutrient solutions or in agar media with a pH indicator. Proton extrusion was enhanced by either iron deficiency, addition of fusicoccin, or single salt solutions of ammonium or potassium salts. The three types of proton extrusion differ in both localization along the roots and capacity. From their sensitivity to ATPase inhibitors it seems justified to characterize them as proton pumps driven by plasma membrane APTases.Enhanced proton extrusion induced by preferential cation uptake from (NH.)2SO4 or K2SO4 was uniformly distributed over the whole root system. In contrast, the enhancement effect of fusicoccin was confied to the basal root zones and that of iron deficiency to the apical root zones. Also the rates of proton extrusion per unit of root fresh weight differed remarkably and increased in the order. Fusicoccin << K2SO4< (NH4hSO4 < iron deficiency.Under iron deficiency the average values of proton extrusion for the whole root system are 5.6 micromoles H' per gram fresh weight per hour, however, for the apical root zones values of about 28 micromoles H can be calculated. This high capacity is most probably related to the iron deficiency-induced formation of rhizodermal transfer cells in the apical root zones. It can be assumed that the various types of root-induced acidification of the rhizosphere are of considerable ecological importance for the plant-soil relationships in general and for mobilization of mineral nutrients from sparingly soluble sources in particular.In higher plants, electrogenic proton pumps are localized in various membranes, such as the plasma membranes, tonoplast or mitochondrial membranes (1-4, 7). It importance. Acidification ofthe soil/root interface (rhizosphere) not only mobilizes mineral nutrients (e.g. P, Fe, Zn; [5,20]) but also affects the activity of microorganisms such as Rhizobium (16) or pathogenic fungi (23,27). This acidification of the rhizosphere can be easily demonstrated by agar techniques and depends on plant species, plant age, form and level of nutrient supply, and buffer capacity of the soil (13). Furthermore, deficiency of phosphorous (8) In this paper we report on results with intact sunflower plants on both the rate of proton extrusion and localization of proton pumps along roots as induced by iron deficiency, FC2 and different nutrient supply. In a following paper the properties of the iron deficiency-induced proton pumps will be described in more detail. MATERIALS AND METHODSGrowth of plants. Seeds of sunflower (Helianthus annuus L. cv Sobrid) were germinated for 3 d in quartz sand moistened with saturated CaS04 solution and subsequently transferred to a continuously aerated nutrient solution (25 plants per 1.5-L plas-

Section: Discussionmentioning

confidence: 80%

“…With one exception (19), all studies on the effect of FC have HW/g fresh weight-h can be calculated for these root zones. These been performed using isolated plant tissue, e.g.…”

Section: Discussionmentioning

confidence: 99%

Localization and Capacity of Proton Pumps in Roots of Intact Sunflower Plants

Römheld¹,

Müller²,

Marschner³

1984

193

“…Fusicoccin, a diterpene glucoside produced by the fungus Fusicoccum amyadali, has been shown to cause hyperpolarization of the membrane potential (4), and to promote K influx and H+ efflux (9,14) possibly by stimulating the exchange of H+ for K+ (2) in a variety of plant tissues. Addition of 0.01 mm fusicoccin stimulated the rate of K+ influx into isolated tobacco protoplasts by more than 200%Yo (Fig.…”

Section: Resultsmentioning

confidence: 99%

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

Mettler¹,

Leonard²

1979

An investigation was conducted into the feasibility of using enzymkally isolated protoplasts from suspension-cultured cells of Nkodana gludnoss L. to study ion transport. Transport of K' (MRb), MC-, H23PO-and "Ca2" from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoli step gradient. Influx of K+, Cl1, and HsPO4-measued over a 30-minute period was reduced (up to 99%) by respiratory inhbitors such as 5 micrograms per militer oligomycin, 0.1 m r niphenoL, 0.1 milimolar cyanide, or N2 gas. In contrast, Ca2' influx was not tightly coupled to respiratory energy production. The influx of K+ was highest between pH 6.5 and 7.5 whereas the influx of H2PO4 and Ca was greatest between pH 4.5 and 5.5. Influx of K+ and Cl was maximal at 35 and 45 C, respectively, and was aimost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K+ influx by more than 200% but had no effect on the influx of either Cl-or Studies on the mechanism of ion transport through the plasma membrane of plant cells are hindered by the presence of the rigid cell wall and the inherent difficulty of working with complex tissues. For example, the cell wall continuum represents a serious physical barrier which prevents direct experimental access to the plasma membrane. Direct access to the plasma membrane would allow new techniques, such as introducing substances into cells with lipid vesicles (16) or membrane labeling to identify plasma membranes during purification (1, 6), to be utilized in the study of ion transport in plants. The development of a simple, well defined, model plant cell system without the interference of the cell wall is needed for new approaches to research on the mechanism of ion transport in plants.Isolated protoplasts (see ref. 3 for review) may provide such a plant cell system. Although there are several studies where transport measurements were made using isolated plant protoplasts (7, 18-20, 22, 24), a detailed analysis of the ion transport properties of isolated protoplasts has not been reported. Such an analysis is needed to evaluate the potential for isolated protoplasts as a model system for studying membrane transport in plants. Research of this kind is also necessary to determine if plasma membrane function has been impaired by protoplast isolation.Here we report an investigation into the feasibility of using protoplasts to study ion transport through plant cell membranes. MATERIALS AND METHODSCeil Culture. Experiments were conducted with cells of Nicotiana glutinosa L. grown in liquid suspension culture according to Uchimiya and Murashige (23). Callus was obtained from stem pith sections and maintained by monthly subculture. To start the suspension cultures, 1 g of callus was placed in a 125-ml Delong flask with 25 ml of nutrient medium (23). The suspension cultures were maintained by subculturing at 1-or 2-week intervals. Suspension cultures were used for protoplast isolations from the 2nd to 12th passa...

“…We cannot as yet say what effect the removal of K+ in the presence of FC has on the outward current, but it has been suggested that the stimulation of K+ influx by FC may be a property of mature nongrowing root tissues (29).…”

Section: Hypothesismentioning

confidence: 99%

“…There would appear, therefore, to be a complex relationship between applied FC and root segment metabolism (including growth). These are complicated further when considering intact roots since it is clear that the growth responses to FC are connected to the age of the root tissues and their morphological characteristics (11,29). Thus, it would appear that the effect of FC does not depend on the increase of cell elongation.…”

Section: Current Generation and Root Growthmentioning

confidence: 99%

Correlation between Root-Generated Ionic Currents, pH, Fusicoccin, Indoleacetic Acid, and Growth of the Primary Root of Zea mays

Miller¹,

Gow²

1989

Correlations between root-generated ionic currents, extracellular pH, indoleacetic acid, fusicoccin, and growth were investigated. Current consistently entered the meristematic and elongating tissues of intact growing roots of Zea mays cv Golden Bantam. Mature root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ionic current was carried by H. Calcium ions did not carry current, but calcium may regulate the proton circulation since the proton current density was slightly larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and an extended zone of inward current. Conversely decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The effect of the fungal toxin fusicoccin was to increase the current density of the inward limb of the ion current and to increase root extension. Concentrations of indoleacetic acid that reduced root growth, also reduced the density of the inward current and shortened the inward current zone. The results emphasize the point that roots are electrically contiguous over many millimeters and that the electrophysiology of root growth is best studied in intact root systems.