Christoph Plieth

Reorientation of Seedlings in the Earth's Gravitational Field Induces Cytosolic Calcium Transients

The gravitational field controls plant growth, morphology, and development. However, the underlying transduction mechanisms are not well understood. Much indirect evidence has implicated the cytoplasmic free calcium concentration ([Ca2+]c) as an important factor, but direct evidence for changes in [Ca2+]c is currently lacking. We now have made measurements of [Ca2+]c in groups of young seedlings of Arabidopsis expressing aequorin in the cytoplasm and reconstituted in vivo with cp-coelenterazine, a synthetic high-affinity luminophore. Distinct [Ca2+]c signaling occurs in response to gravistimulation with kinetics very different from [Ca2+]c transients evoked by other mechanical stimuli (e.g. movement and wind). [Ca2+]cchanges produced in response to gravistimulation are transient but with a duration of many minutes and dependent on stimulus strength (i.e. the angle of displacement). The auxin transport blockers 2,3,5-tri-iodo benzoic acid and N-(1-naphthyl) phthalamic acid interfere with gravi-induced [Ca2+]cresponses and addition of methyl indole-3-acetic acid to whole seedlings induces long-lived [Ca2+]ctransients, suggesting that changes in auxin transport may interact with [Ca2+]c. Permanent nonaxial rotation of seedlings on a two-dimensional clinostat, however, produced a sustained elevation of the [Ca2+]c level. This probably reflects permanent displacement of gravity-sensing cellular components and/or disturbance of cytoskeletal tension. It is concluded that [Ca2+]c is part of the gravity transduction mechanism in young Arabidopsis seedlings.

Self-Reporting Arabidopsis Expressing pH and [Ca2+] Indicators Unveil Ion Dynamics in the Cytoplasm and in the Apoplast under Abiotic Stress

For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca2+]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca2+] in the apoplast and in the cytoplasm were studied under salt and “drought” (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells.

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

SummaryThis review focusses on Ca2+-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.

A novel fluorescent pH probe for expression in plants

BackgroundThe pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av- GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt- GFP) and compare its properties with pHluorins for expression and use in plants.ResultspHluorins have a higher pH-sensitivity. However, Pt- GFP has a broader pH-responsiveness, an excellent dynamic ratio range and a better acid stability. We demonstrate how Pt-GFP expressing Arabidopsis thaliana report cytosolic pH-clamp and changes of cytosolic pH in the response to anoxia and salt-stress.ConclusionPt- GFP appears to be the better choice when used for in vivo- recording of cellular pH in plants.

Temperature Sensing by Plants: Calcium-Permeable Channels as Primary Sensors—A Model

Abstract. 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., [Ca2+]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 [Ca2+]c changes well. This suggests that the primary temperature sensor in plants might be a Ca2+-permeable channel.

Cytoplasmic Ca2+-H+-exchange buffers in green algae

SummaryFluorescence ratio imaging was used for simultaneous measurement of cytosolic pHc and pCac inChara corallina, Nitella flexilis, andEremosphaera viridis. In some experiments the electrical membrane potential was also recorded. The first hint of coupling between changes in pHc and pCac was found in characean cells when the influence of butyrate on cytosolic streaming was studied by laser-Doppler-anemometry (LDA). The observed butyrate-induced cessation of cytosolic streaming supports the assumption that changes in pHc cause changes in pCac. This hypothesis was tested by simultaneously loading cells with Fura-2-dextran and BCECF-dextran. The addition of butyrate revealed strong coupling between pCac and pHc although this only occurred when the difference between pHc and pCac was less than 0.4 units (± 0.24, n=7). The measured relationship between the changes in pCac and pHc could be fitted by a cytoplasmic buffer exchange process. Protons imported by butyrate into the cytoplasm are able to displace Ca++ ions from cytoplasmic buffer sites. Three dissociation constants of the cytoplasmic buffer were pK1=6.2, pK2=7.1 for proton buffering, and pKca=6.7 for Ca++ ion buffering. Other possible mechanisms, such as butyrate-induced Ca++ influx through the plasmalemma and Ca++ release from internal stores are discussed. They are not necessary to explain the observed coupling but cannot be excluded from the process. Using the butyrate technique, the cytosolic in vivo proton buffer capacities ofN. flexilis, C. corallina, andE. viridis were determined as βi=30 mM · H+/pH-unit, βi=46 mM · H+/pH-unit, and βi=90 mM · H+/pH-unit, respectively. The values obtained in vivo are greater than those found previously using extraction methods. This can be explained in terms of pump activity and exchange with cell organelles, i.e., the vacuole. The high value of βi found inEremosphaera reflects adaptation to its habitat.

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 Ca2+ into the chloroplasts.

Aequorin as a Reporter Gene

Reporter proteins allow one to monitor cellular parameters that are involved in signal transduction, development, metabolic processes, and transport. There are targeting strategies available to direct the indicator protein exactly to the locale inside the organism from which information is desired. This circumvents experimental reductionism and allows experimentation with whole intact and undisturbed organisms. The outstanding advantages of self-reporting organisms make it worth to shoulder cost- and time-consuming molecular work. Here, the luminescent Ca2+ indicator aequorin is introduced and a rough guideline is given from early planning the molecular work and assembling an experimental setup to experimentation with luminescent Arabidopsis, data processing, and control experiments.

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K+ channel as studied by a kinetic analysis in Chara corallina

Abstract. The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca2+]c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca2+]c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K+ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K+ transport of the plasma membrane: (i) light causes an uptake of Ca2+ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca2+]c, (iii) this leads to a decrease in the activity of a K+ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca2+ fluxes coming out of the chloroplasts upon darkening cause a Ca2+-induced phosphorylation of myosin, which slows down cytoplasmic streaming.