Bert van Duijn

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as α-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 μl), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.

The presence and subcellular localization of caspase 3‐like proteinases in plant cells

Caspases play a very important role in initiating and executing apoptotic processes in animal cells. In this study we show that plant mitochondria were able to initiate the activation of caspase 3 in a Xenopus cell free system. Caspase 3‐like activity was found to be present in plant cells and could only be inhibited by the specific caspase 3 inhibitor N‐acetyl‐Asp‐Glu‐Val‐Asp‐fluoromethylketone (Ac‐DEVD‐fmk) and not by cysteine protease inhibitors. By micro‐injection of the caspase 3 substrate in living Chara cells we showed that caspase 3‐like activity was mainly present in the cytosol rather than in the vacuole. This is the first time that in vivo caspase 3‐like activity has been demonstrated in plants.

Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts

Verapamil, nifedipine and bepridil, three antagonists of L‐type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacumcv. Xanthi protoplasts with IC50 of 5 μM, 5 μM and 1 μM, respectively. In the outside‐out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.

Abscisic acid induces a cytosolic calcium decrease in barley aleurone protoplasts

Cytosolic calcium concentrations (Ca1) of barley aleurone protoplasts after stimulation with the plant hormone abscisic acid (ABA) were measured by using the calcium‐sensitive flourescent dye Indo.1. The measured basal Ca1 is about 200 nM. Stimulation with ABA induces a strong dose‐dependent decrease in Ca1 to a minimal value of about 50nM. The decrease occurs within 5 s. The Ca2+ antagonists La3+ and Cd2+ inhibit the ABA‐induced Ca1 decrease in a dose‐dependent manner, while the Ca2+ channel blockers verapamil and nifedipine give no inhibition. The induction of Ca1 decrease by ABA is consistent with activation of the plasma membrane Ca2+‐ ATPase by ABA. The possible role of this ABA‐induced Ca1 decrease in ABA signal transduction and in counteracting the effects of gibberellic acid are discussed.

Intracellular K+, Na+ and Cl− concentrations and membrane potential in human moncytes

The relationship between the resting membrane potential and the intracellular ionic concentrations in human monocytes was investigated. Cell volume, cell water content, and amount of intracellular K+, Na+, and Cl- were measured to determine the intracellular concentrations of K+ (Ki), Na+ (Nai) and Cl- (Cli) of monocytes, and of lymphocytes and neutrophils. Values found for monocytes were similar to those for neutrophils, i.e., cell volumes were 346 and 345 micron3, respectively, cell water content 78%, and Ki, 128 and 125, Nai, 24 and 26, and Cli, 102 and 103 mmol/l cell water, respectively. Lymphocytes, however, had different values: 181 micron3 cell volume, 77% cell water content, and for Ki, Nai, and Cli, 165, 37, and 91 mmol/l cell water, respectively. The resting membrane potential of cultured human monocytes (range -30 to -40 mV), determined by measurement of the peak potential occurring within the first milliseconds after microelectrode entry, was most dependent on extracellular K+, followed by Cl-, and Na+. The membrane permeability ratio of Cl- to K+ was estimated by use of the constant field equation to be 0.23 (range 0.22 to 0.30).

Hodgkin-Huxley analysis of whole-cell outward rectifying K+-currents in protoplasts from tobacco cell suspension cultures

SummaryThe voltage and time dependence of outward-rectifying K+ currents (Ik,out) measured in protoplasts from tobacco cell suspension cultures in the whole-cell configuration of the patch-clamp technique are quantitatively analyzed. The voltage and time dependence was described according to the Hodgkin and Huxley model for Ik,out currents in the squid giant axon, and to allow comparison, in analogy with the quantitative analysis of IK,out currents in Vicia faba guard cell protoplasts as described by Schroeder (J. Membrane Biol., 107:229–235, 1989). The IK,out, from tobacco could be described by a similar model as the IK,out from guard cell protoplasts (i.e., sigmoid activation time course, activation variable raised to second power, single exponential deactivating tail currents, absence of inactivation). However, in contrast to guard cells, both the activation and deactivation time constants were strongly voltage dependent in tobacco protoplasts. The voltage dependence of the transition rates for channel opening and channel closing was slightly asymmetrical and inverse to the asymmetry found in guard cells. The data presented show that the voltage-dependent kinetic properties of the IK,out conductance of tobacco protoplasts are different from these properties in guard cell protoplasts. This analysis provides a basis for the study of IK,out conductance function and modulation.

The membrane potential of the cellular slime mold Dictyostelium discoideum is mainly generated by an electrogenic proton pump

Trans membrane potential or ionic current changes may play a role in signal transduction and differentiation in the cellular slime mold dictyostelium discoideum. Therefore, the contribution of electrogenic ion pumps to the membrane potential of D. discoideum cells was investigated. the (negative) peak-value of the rapid potential transient, seen upon microelectrode impalement, was used to detect membrane potential changes upon changes in the external pH in the range of 5.5 to 8.0. The membrane potential was close to the Nernstian potential for protons over the pH range 5.5 to 7.5. The acid-induced changes in membrane potential were consistent with outward-proton pumping. The maximal membrane potential was at pH 7.5. Furthermore, the proton pump inhibitors diethylstilbestrol, miconazole and zearalenone directly depolarize the membrane. Cyanide and temperature decrease cause membrane depolarization as well. During recovery from cyanide poisoning a H+ efflux is present. From these measurements we conclude that the membrane potential of d. discoideum cells is mainly generated by an electrogenic proton pump. Measurements in cells with different extracellular potassium and H+ concentrations suggest a role for potassium in the function of the electrogenic proton pump. These results provide a framework for future research towards a possible role for the proton pump in signal transduction and differentiation.

Electrophysiological properties of Dictyostelium derived from membrane potential measurements with microelectrodes.

SummaryElectrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueEp (−19 mV) should be used as an indicator of the resting membrane potentialEm ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueEn (−5 mV). Using enlarged cells such as giant mutant cells (Ep=−39 mV) and electrofused normal cells (Ep=−30 mV) improved the reliability ofEp as an indicator ofEm. From the data we concluded thatEm ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl2 is at least −50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceRi of the impaled cells was 56 MΩ for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 GΩ. Specific membrane capacitance was 1–3 pF/cm2. Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inRi of the impaled cell. It was followed by a depolarization, which was associated with a decrease inRi. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.

Intracellular microelectrode membrane potential measurements in tobacco cell-suspension protoplasts and barley aleurone protoplasts: interpretation and artifacts.

Intracellular microelectrode measurements in plant cell protoplasts have been widely used to study hormone signal transduction processes. However, the interpretation and reliability of such measurements are largely dependent on a detailed evaluation of the measurement conditions, as investigated in the present paper. Upon microelectrode penetration of tobacco cell suspension protoplasts and of barley aleurone protoplasts a fast negative going impalement-induced potential transient of less than a few ms duration could be observed. After reaching a steady-state potential at the ms time scale the measured potential hyperpolarized again and, in most cases, subsequently depolarized to a new steady-state value. Analysis of the electrical equivalent circuit of the measurement configuration showed that the occurrence of the impalement-induced potential transient indicates that these measurements suffer from a microelectrode-induced shunt resistance which loads the measurement. In addition, it is shown that the peak-value of the potential transient is the most reliable indicator of the true membrane potential and of true membrane potential changes of the protoplast, since this value is rather membrane resistance independent. For correct interpretation of steady-state measurements of membrane potential and stimulus-induced membrane potential changes data on membrane and shunt resistance are essential. As an example of the measurement of membrane potential changes the effects of 1-NAA on measured potential values in tobacco protoplasts and the effect of extracellular pH changes on barley aleurone protoplasts are analyzed with regard to the above described conclusions.