Cold-induced cytosolic free calcium ion concentration changes in wheat.

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca(2+)](c)) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca(2+)](c) was detected in vivo by luminometry, and [Ca(2+)](c) elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca(2+)](c) during cold exposure. Temperature-induced changes in [Ca(2+)](c) were found to be more dependent on the change in temperature (dT dt(-1)) than on the absolute value of temperature. [Ca(2+)](c) increased only at cooling rates higher than 8 degrees Cmin(-1), indicating that an overall cellular [Ca(2+)](c) increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called 'calcium signature hypothesis'.

Effect of CO2 supply on formation of reactive oxygen species in Arabidopsis thaliana.

Light-induced generation of reactive oxygen species (ROS) in 2-week-old leaves of Arabidopsis thaliana was studied by means of the ROS-sensitive dyes nitroblue tetrazolium (NBT) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Superposition of pictures of chlorophyll fluorescence and DCF fluorescence indicated that the origin of ROS was in the chloroplasts. Experiments were done with zero, 0.1, or 10 mM NaHCO3 in the infiltration medium. Energy quenching in photosystem II was higher under low CO2 concentrations as measured by chlorophyll fluorescence. DCF fluorescence showed that CO2 deficiency led to an increase of ROS generation. In contrast, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced the light-induced increase of DCF fluorescence. This indicates that ROS production does not primarily result from over-reduction of photosystem II as caused by impeding electron flow in the electron transfer chain. More likely, it is an effect of diverting electron flux normally aimed at carboxylation in the Calvin cycle to other sinks more prone to the generation of toxic radicals. There was no significant effect of salicyl hydroxamate (a blocker of the alternative oxidase), showing that the mitochondrial electron transfer chain seems to play a minor role as already indicated by the superposition of chlorophyll and DCF fluorescence.

Light-induced decrease in DCF fluorescence of wheat leaves in the presence of salicyl hydroxamate.

5-(and-6)-Carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), a permeative indicator of oxidative stress, was loaded into dissected leaves of wheat in order to monitor the temporal development of reactive oxygen species. DCF fluorescence was found to be constant under dark conditions. Upon loading the leaves with salicyl hydroxamate, a blocker of the alternative oxidase, DCF fluorescence linearly increased in the dark. This indicates a function of alternative oxidase in preventing reactive oxygen radicals in the mitochondria. Upon illumination, the DCF signal decreased within 5 min. As illuminated chloroplasts would increase the load of reactive oxygen species, the observed decrease cannot be assigned to the production of reactive oxygen species in the chloroplasts. Three different putative mechanisms are considered which all assign an important role to light-induced delivery of NAD(P)H: (1) direct quenching of DCF fluorescence by light-generated NAD(P)H, (2) light-stimulated activation of scavenging enzymes, or (3) redirection of mitochondrial electron fluxes as caused by the delivery of excess redox equivalents (NADH) from the chloroplasts.

Plant calcium signaling and monitoring: pros and cons and recent experimental approaches.

This review focusses on Ca(2+)-mediated plant cell signaling and optical methods for in vivo [Ca2+] monitoring and imaging in plants. The cytosolic free calcium concentration has long been considered the central cellular key in plants. However, more and more data are turning up that critically question this view. Conflicting arguments show that there are still many open questions. One conclusion is that the cytosolic free Ca2+ concentration is just one of many cellular network parameters orchestrating complex cellular signaling. Novel experimental strategies which unveil interference of cellular parameters and communication of transduction pathways are required to understand this network. To date only optical methods are able to provide both kinetic and spatial information about cellular key parameters simultaneously. Focussing on calcium there are currently three classes of calcium indicators employed (i.e., chemical fluorescent dyes, luminescent indicators, and green-fluorescent-protein-based indicators). Properties and capabilities as well as advantages and disadvantages of these indicators when used in plant systems are discussed. Finally, general experimental strategies are mentioned which are able to answer open questions raised here.

Temperature sensing by plants: calcium-permeable channels as primary sensors--a model.

Recently the properties of temperature sensing in plants have been demonstrated experimentally by Plieth et al. (The Plant Journal 1999. 18:491-497). The relevant biophysical parameters are established here by mathematical modeling in order to understand the experimental findings in quantitative terms. A simple one-compartment model is presented, as a preliminary approach to explain how the input signal (i.e., temperature T) is perceived and how the information is translated into an output signal in the plant cell (i.e., [Ca(2+)](c)). The model is based on the fact that calcium influx into the cytoplasm is mediated by calcium-permeable channels which are assumed to be solely dependent on cooling rate (dT/dt) and calcium efflux is mediated by calcium pumps which have been shown to be dependent on absolute temperature (T). Firstly, it is demonstrated that this model is able to meet the demand for a satisfactory interpretation of the experimental data, and secondly that it reproduces the experimentally observed features of the cooling induced [Ca(2+)](c) changes well. This suggests that the primary temperature sensor in plants might be a Ca(2+)-permeable channel.

Temperature sensing by plants: the primary characteristics of signal perception and calcium response.

Cold elicits an immediate rise in the cytosolic free calcium concentration ([Ca2+]c) of plant cells. We have studied the concerted action of the three underlying mechanisms, namely sensing, sensitisation and desensitisation, which become important when plants in the field are subjected to changes in temperature. We applied different regimes of temperature changes with well-defined cooling rates to intact roots of Arabidopsis thaliana expressing the calcium-indicator, aequorin. Our results indicate that temperature sensing is mainly dependent on the cooling rate, dT/dt, whereas the absolute temperature T is of less importance. Arabidopsis roots were found to be sensitive to cooling rates of less than dT/dt = 0.01 degrees C/s. However, at cooling rates below 0.003 degrees C/s (i.e. cooling 10 degrees C in 1 h) there is no detectable [Ca2+]c response at all. At low temperature, the sensitivity of the plant cold-detection system is increased. This in turn produces greater cooling-induced [Ca2+]c elevations. Prolonged or repeated cold treatment attenuates the [Ca2+]c responses to subsequent episodes of cooling.

Low-pH-mediated elevations in cytosolic calcium are inhibited by aluminium: a potential mechanism for aluminium toxicity.

Aluminium, the most abundant metal in the earth's crust, is highly toxic to most plant species. One of the prevailing dogmas is that aluminium exerts this effect by disrupting cellular calcium homeostasis. However, recent research gives strongly conflicting results: aluminium was shown to provoke either an increase or a decrease in cytosolic free calcium concentration ([Ca2+]c). To solve this question, we have adopted a novel approach: [Ca2+]c measurements in intact plant roots as opposed to isolated cells, and the correlative measurements of intracellular and external pH. The results obtained show that plant roots respond to low external pH by a sustained elevation in [Ca2+]c. In the presence of aluminium, this pH-mediated elevation in [Ca2+]c does not occur, therefore any potential calcium-mediated protection against low pH is likely to be irreversibly inhibited. The severity of the inhibitory effect of aluminium on [Ca2+]c depends on the concentration of external calcium, thus perhaps explaining why the effects of aluminium toxicity are ameliorated in calcium-rich soils. It seems possible that a primary toxic effect of aluminium might be to impair calcium-mediated plant defence responses against low pH.

Strontium-induced repetitive calcium spikes in a unicellular green alga

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

Repetitive Ca2+ spikes in a unicellular green alga.

Cytosolic Ca2+ activity ([Ca2+]cy) and membrane potential were measured simultaneously in the unicellular green alga Eremosphaera viridis. Steady state [Ca2+]cy was about 160 nM. A 'light-off' stimulus induced a transient elevation of [Ca2+]cy ([Ca2+]cy spike) in parallel with a transient hyperpolarization of the plasma membrane. Caffeine and Sr2+, known to release Ca2+ from intracellular stores in animal cells, induced repetitive [Ca2+]cy spikes in Eremosphaera which were always accompanied by parallel repetitive transient hyperpolarizations. These transient hyperpolarizations could be used as an indicator for [Ca2+]cy spikes. Repetitive [Ca2+]cy spikes in Eremosphaera were similar to repetitive [Ca2+]cy spikes in excitable animal cells. The mechanisms underlying these [Ca2+]cy oscillations seem to be comparable in animal and plant cells.

Ion channel activity during the action potential in Chara: new insights with new techniques.

The dynamics of macroscopic currents underlying the electrically triggered action potential (AP) in the giant alga Chara corallina were directly recorded with an action potential clamp method. In this technique an AP is recorded and repetitively replayed as the command voltage to the same cell under voltage control. Upon adding the channel blockers niflumic acid and/or Ba(2+) to the bath, the excitation current, i.e. the current crossing the membrane during an AP, can be dissected into a transient, fast-appearing Cl(-) inward current and a transient delayed K(+) outward current. The delayed onset of the K(+) outward current demands the postulation of an additional outward current in order to balance the excess Cl(-) inward current at the onset of the AP. The capacitive current that alters the charge on the membrane during excitation is several orders of magnitude too small to be relevant for charge balance. Measurements of single channel activity in the plasma membrane of C. corallina by the patch clamp method shows two types of Cl(-) channel (15 and 38 pS with 100 mM Cl(-) in the pipette) and one type of K(+) channel (about 40 pS with 100 mM K(+) in the pipette) which become transiently active during an AP. Typically, variable numbers of CI(-) channels activate in a random fashion for short periods of time when favoured by positive voltages in combination with high concentrations of extracellular Ca(2+) (Ca(2+)(o)) or during an AP of the whole cell. The peak values of these Cl(-) channel currents measured in a patch are such that they can account quantitatively for the peak of the whole cell Cl(-) excitation current studied under comparable ionic conditions. Furthermore, the short dura- tion of channel activity, as well as the fast rising and somewhat slower trailing kinetics is similar in duration and dynamics to AP-associated changes in membrane permeability of the whole Chara cell to Cl(-) (P(Cl(-))). Taken together, the data stress that the characteristic, transient activation of random numbers of Cl(-) channels seen in membrane patches is the elementary unit of the Cl(-) excitation current. However, due to the random nature of this transient activity, gating of Cl(-) channels can not be explained on the basis of previous models for excitation: gating can neither be due to intrinsic voltage sensitivity of the Cl(-) channels, nor to a voltage-dependent influx of Ca(2+) and subsequent activation of Ca(2+)-sensitive Cl(-) channels. To account for the short life-time and for the randomness of Cl(-) channel activity, the putative gating factors Ca(2+) and voltage must be uncoupled in time. This could be explained by a random release of Ca(2+) from stores, the latter being filled in a voltage-sensitive manner via non-specific cation channels from the outside. A 4 pS non-selective cation channel in the plasma membrane may serve this purpose. The 40 pS K(+) channel, which becomes transiently active in C. corallina during a cell AP, is an outward rectifier. At negative resting voltages the channel has a low open probability (< <1%). At voltages reached during an AP the open probability rises significantly reaching half-maximal open probability at -25 mV. The elevated activity of the 40 pS channel associated with membrane excitation relaxes at the end of an AP with a time constant of about 2.5 s. A comparable time constant of 2 s can be obtained for the decay of the transiently elevated permeability of the membrane to K(+) (P(K(+))), stressing that the kinetic properties of the 40 pS K(+) channel are responsible for the course of whole cell P(K(+)) changes. Voltage sensitivity of the K(+) channels suggests that they are activated during an AP by the drop in membrane voltage in order to aid repolarization. However, the rise and decay of P(K(+)) during an AP also shares similarity with the time-course of transient changes in cytoplasmic concentration of free Ca(2+), [Ca(2+)](cyt), the latter being measured in parallel experiments with the Ca(2+)-sensitive fluorescent dye, Fura-2, in excited C. corallina cells. This similarity could suggest that gating of the 40 pS K(+) channel is also sensitive to [Ca(2+)](cyt) and that the latter sensitivity is rate-limiting for activity during an AP.

Light dependence of protoplasmic streaming in Nitella flexilis L. as measured by means of laser-velocimetry.

Laser-velocimetry was applied in order to study the effect of light on the velocity of protoplasmic streaming (pps) in Characean cells. A change from dark to light (= 6 W · m(-2)) leads to an acceleration of streaming by about 15-30% with a time-constant of approx. 300 s. The transition from light to dark causes a transient decrease of velocity below the original dark level. This response occurs with a time constant of about 500 s. It returns to its initial value with a time-constant of about 2000 s. This may indicate that a control loop of cytosolic homeostasis takes a decrease in pCa more seriously than an increase. A possible involvement of temperature effects caused by illumination was excluded by measuring the influence of temperature. Steady-state velocity of streaming changed by 5% per 1° C. Irradiation with infra-red light (λ > 780 nm) did not cause a change in velocity. The absence of a light effect on streaming velocity in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) shows that photosynthesis and not phytochrome is involved. The role of light-induced changes of pCa is discussed, especially with respect to the hypothesis of Vanselow and Hansen (1989, J. Membr. Biol. 110, 175-187) that photosynthesis acts on the plasmalemma K(+)-channel via light-induced uptake of Ca(2+) into the chloroplasts.

In vitro activity of flomoxef in comparison to other cephalosporins.

Flomoxef and cefazolin had nearly the same activity against staphylococci, which was stronger than that of other cephalosporins. Against Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus pneumoniae, cefotaxime and cefazolin were more active than flomoxef, but the other cephamycins were less active than flomoxef. In comparison to the other cephalosporins, latamoxef and flomoxef had higher activity against Branhamella catarrhalis, whereas cefotaxime, latamoxef and cefotetan were more active against Haemophilus influenzae. Flomoxef was the only drug exhibiting activity against Clostridium difficile. The activity of flomoxef and latamoxef against Bacteroides fragilis was stronger than that of the other cephalosporins, but Bacteroides bivius was resistant to each of these antibiotics.