15 research outputs found

    The role of the plasma-membrane Ca2+-ATPase in ca2+ homeostasis in Sinapis alba root hairs

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    The regulation of cytosolic Ca2+ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca2+-ATPase. For this purpose, erythrosin B was used to inhibit the Ca2+-ATPase, and the Ca2+ ionophore A23187 was applied to manipulate cytosolic free [Ca2+] which was then measured with Ca2+-selective microelectrodes. (i) At 0.01 M, A23187 had no effect on the membrane potential but enhanced the Ca2+ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca2+ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca2+ spontaneously reversed and became an increase, a process which strongly depended on the external Ca2+ concentration, (iv) Upon removal of A23187, the cytosolic free [Ca2+] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca2+ is an activation of Ca2+ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca2+ from the cytosol. The two observations that after the addition of A23187, (i) Ca2+ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca2+-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH+/Ca2+-ATPase. Based on the finding that the Ca2+-ATPase inhibitor erythrosin B had no effect on cytosolic Ca2+, but caused a strong Ca2+ increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca2+-pump. We further conclude that this ATPase is a major Ca2+ regulator in stress situations where the cytosolic Ca2+ has been shifted from its steady-state level, as may be the case during processes of signal transduction

    Signal Transduction in Sinapis alba Root Hairs: Auxins as External Messengers

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    In developing root hairs of Sinapis alba the effects of externally applied indole-3-acetic acid (IAA) and other auxins have been investigated with respect to membrane potential, membrane conductance, cytosolic Ca2 + and pH. Following a delay of roughly 30s, 10- 12 to lO- IO M IAA slowly hyperpolarize, 10- 7 M IAA rapidly depolarize the root hairs, while 10- 9 M has hardly an effect. We show that these voltage responses are not the result of a change in membrane conductance or permeability, but are presumably caused by a change in H+ ATPase activity. The other tested auxins and analogues yielded comparable effects, but with much lower effectivity (IAA > 1-NAA > 2,4-D ~ 2-NAA > 2,3-D). Cytosolic Ca2+ and pH were decreased during depolarization by 0.2 and 0.4 units, respectively. No such changes were observed during hyperpolarization or about 1 h after the first encounter of the root hairs with IAA. We propose that IAA is a natural external signal for roots while competing with neighboring organisms for nutrients and salts, and suggest a signal chain with the plasma membrane H+ ATPase as a target protein. The delay in response to IAA, the time dependency, and the extremely low effective IAA concentrations point to the existence of a IAA receptor. Since the IAA-induced shifts in cytosolic pH and Ca2+ occur simultaneously with the depolarization, the question whether these ions are cellular messengers and part of an IAA-triggered signal chain is critically discussed

    Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

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    Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H+ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2–4 h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of −152±5 mV, hyphae of B. graminis yield −251±8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a “light-off” stimulus. Five days after inoculation, apoplastic pH has increased to 5.97±0.47 (n=11) and does no longer respond to “light-off” when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to “light-off”. It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility