Ekkehard Schönbohm

No evidence for Ca2+ influx as an essential link in the signal transduction chains of either light-oriented chloroplast movements or Pfr-mediated chloroplast anchorage in Mougeotia

The effect of Ca2+ entry blockers on the light-induced chloroplast movements in Mougeotia was studied. The organic inhibitors diltiazem, nifedipine and ruthenium-red and the inorganic inhibitors La3+ and Co2+ do not affect the low or high fluence rate movements of chloroplasts. Only at toxic concentrations or after long-term incubations (4 – 7 days), which partially damage the cells, is chloroplast movement slightly inhibited. The Pfr-mediated increase in chloroplast anchorage and the chloroplast anchorage of dark-sensitized algae are not significantly altered by Ca2+ entry blockers within physiological concentrations. Our results contradict the hypothesis of a Pfr-controlled Ca2+ influx as an essential link in the signal transduction chain of light-oriented chloroplast movements in Mougeotia.

Die wirkung von SH-blockern sowie von licht und dunkel auf die verankerung der Mougeotia-chloroplasten im cytoplasmatischen wandbelag: 2. Mitteilung: Zur mechanik der chloroplastenbewegung

Summary Chloroplasts of Mougeotia are moving light-oriented in the cell. As a hypothesis, it is supposed that contractile fibrils (or microtubles?) are active in the mechanism of chloroplast movement. It then would be possible that the postulated cytoplasmic elements are contractile ATP-ases being sensitive to blockers of SH-groups. In the following experiments the effect of SH-blockers on chloroplast movement as well as on fastening chloroplasts to cytoplasmic parietal coating (= «cpc») will be tested; this fastening is also investigated as influenced by light and darkness. For measurement of fastening the plastids to «cpc» we make use of the centrifugation method (fig. 2). 1. The effect of SH-blockers on light-oriented chloroplast movement of Mougeotia . Light-oriented movements of Mougeotia -chloroplasts are considerably inhibited by SH-blockers (PCMB, Salyrgan, HgCl 2 ) applicated in non-toxic molarities (fig. 3). This inhibition of the movement can be reversed by cysteine, but only to a small extent by serine and not at all by ATP (fig. 4, tab. 1 and 2). 2. The effect of some external factors (SH-blockers, light, darkness) on fastening chloroplasts to cytoplasmic parietal coating (= «cpc»). a) Fastening chloroplasts to «cpc» is weakend considerably by incubating Mougeotia with SH-blockers (PCMB or Salyrgan: fig. 5). This effect can be reversed by cysteine but not significantly by serine (fig. 5). b) Chloroplasts of cells being highly-sensitized (by pretreatment with darkness or with far-red light!) for a following high intensity movement are fastened to «cpc» to a relatively small extent only, whereas chloroplasts of cells desensitized (by irradiation with weak red or white light!) are fastened to «cpc» to a high extent (figs 6 and 7). Short wave polarized light of high intensities (e.g. 50 kerg/cm2 · sec) weakens fastening of chloroplasts to «cpc» to a high extent (fig. 8). Experimenting in high-intensity-range of polarized short wave light (e.g. 50 kerg/cm 2 · sec) no dichroism for this effect has been obtained, whereas dichroism was found if irradiating Mougeotia with polarized short wave light of lower «high» intensities (e.g. 12 kerg/cm 2 · sec: fig. 8). c) The process of fastening chloroplasts of Mougeotia -cells to a relatively high extent to «cpc» by weak light could be inhibited by blockers of SH-groups considerably. The results of these experiments support the hypothesis that contractile proteins of high sensitivity to SH-blockers are active in the mechanism of light oriented chloroplast movement of Mougeotia . The effect of darkness and of long wave light (red and far-red) on the extent of fastening chloroplasts to «cpc» are discussed in relation to phytochrome-controlled rapid-reactions in higher plants.

ON THE SIGNAL-TRANSDUCTION CHAINS OF TWO Pfr- MEDIATED SHORT-TERM PROCESSES: INCREASE OF ANCHORAGE and MOVEMENT OF Mougeotia CHLOROPLASTS

Abstract— We examined two published hypotheses on the signal‐transduction chain of the light‐oriented chloroplast movements in the fresh‐water alga Mougeotia. One hypothesis postulates a Ca2+‐influx controlled by a tetrapolar gradient of phytochrome in its far‐red light absorbing form (Pfr). The other hypothesis postulates anchorage sites for actin‐filaments even at those areas of the plasmalemma where phytochrome is in its inactive form (Pr). Calmodulin and Ca2+‐sequestering vesicles are assumed to be essential links of this transduction chain.

Biophenole: Steuernde Faktoren bei der lichtorientierten Chloroplastenbewegung?

Summary In an analysis of the role of phenolic compounds 1 ) for the light-oriented chloroplast movement in Mougeotia the following results were obtained: 1. The size of cytosomes (“tannin vesicles”) changes inversely proportional to the amount of the phenolic compounds in the efflux solution. 2. The efflux of phenolic compounds becomes extremely increased by application of ammonium oxalate. 3. The size of cytosomes increases by application of bioflavonoids and concentrated efflux solutions. 4. A relative constant ratio exists between the efflux rates of phenolic compounds and of calcium. 5. 5. A high amount of the total phenolic compounds (efflux and extract!) comes from mobile cytosomes (“tannin vesicles”). 6. 6. In a first crude separation-process (by molecular sieve chromatography) 6 fractions of a phenolextract were received; some of the fractions are strong inhibitors of the high-intensity light but not of the low-intensity light chloroplast movement. 7. Concentrated solutions of the phenol efflux as well as of the phenol extract inhibit chloroplast movement as well as the luciferin/luciferase reaction in experiments to determine the ATP concentration in an extract. These results encourage us to trace the hypothesis on the regulating effect of phenolic compounds in light-oriented chloroplast movements in Mougeotia.

Untersuchungen zum Photoreceptorproblem beim tonischen Blaulicht-Effekt der Starklichtbewegung des Mougeotia-Chloroplasten*

Summary If irradiated simultaneously from different directions with blue light of high intensity and with red light of low intensity, the chloroplasts of Mougeotia are oriented by red rather than by short wave length light. The main effect of blue light seems to be, in this case, to change the chloropla sts from a positive to a negative phototact ic response, oriented by phytochrome. Thus, the additional given blue light has a tonic effect, i. e. independent of its direction (SCHONBOHM, 1966 b, 1967 a). It seems to be possible, that the difference between the action spectra of the high intensity movement of Mougeotia-chloroplasts (SCHONBOHM, 1963) and the action spectra of chloroplast movements of other objects (HAUPT, 1963; HAUPT and SCHONBOHM, 1970) is caused by the fact that both photoperception in phytochrome and photoperception in a yellow pigment arc represented by the action spectra of chloroplast movement of Mougeotia obtained when irradiating only with a single radiation source. In the following experiments we will record action spectra for the tonic blue light effect only; we have to take care of the action-dichroism, which we have found in former investigations (SCHCNBOHM, 1968). Polarized red light vibrating perpendicularly (r ⊥ or parallel (r ∥) to the cell axis is given simultaneously with blue light (bl). The following results have been obtained in the present investigations. 1. a) The action spectrum for the tonic effect of unpolarized blue light (bl *. with r ⊥ does not differ remarkably from the action spectra for the high intensity movement induced by blue light only (ef. fig . 2 with fig. 1). b) If we compare the action spectra for the effect of unpolarized blue light (bl *. with or without r ⊥ with the action spectra for the tonic effect of polarized blue light (bl ∥. with r ⊥) in the latter we can see a pl ateau reaching from ca. 450 nm to 470 nm instead of one peak near 465 nm (fig. 4). In the latter action spectra we do not find a relativel y high peak in the near ultraviolet as we did in the former action spectra (bl *, with or without r ⊥). 2. a) It has been demonstrated that polarized blue light (λ : ca. 400-500 nm) is tonically much more effective, if vibrating parallel to the cell axis (bi ∥, with r ⊥ than if vibrating perpendicularly (bl ⊥, wirh r ⊥) (fig. 3). b) This action dichroism is inverse, if Mougeotia is irradiated with ultraviolet light (λ = 363 nm) rather than with polarized visible blue light (λ > 400 nm) (fig. 7). c) If the wavelength of the blue light is longer than 400 nm the action dichroism for the tonic blue light effect remains constant during the whole day, whereas it is changing in a diurnal rhythm in near ultraviolet light (fig. 8). d) Action dichroism for the tonic blue light effect of the high intensity movement of Mougeotia-chloroplasts cannot be caused by an action dichroism of a countercurrent «low intensity movement» which is induced simultaneously by the same intensities of polarized blue light as are needed for high intensity movement (ef. fig. 9 and 10).

Durch Phytochrom aktivierbare kontraktile Plasmaelemente: Lokalisation in cler Mougeotia-Zelle 5. Mitteilung zur Mechanik cler Chloroplastenbewegung

Summary In former experiments it had been shown that low intensity white or red light increases the extent of fastening chloroplasts to the parietal cytoplasmic layer. This effect is much more pronounced if the E-vector is vibrating perpendicularly rather than parallel to the long axis of the cell (Schonbohm, 1972, 1973 a, b). In a model we suppose that this P fr -egulated increase of fastening chloroplasts is controlled by phytochrome, which is localized in the corticalplasm of the transverse walls (fig. 1). If this model were correct, red light induced fastening chloroplasts should not be realized in cells in which chloroplasts do not contact the cytoplasmic parietal coating of the transverse walls. In contrast to this model the experiments (fig. 2) show that Pfr-mediated increase of fastening chloroplasts even becomes realized in such cells, the chloroplasts of which have been removed from the centripetal transverse walls by pre-centrifugation before red light is given. Thus we have to suppose that red light controlled increase of fastening Mougeotia chloroplasts becomes realized by contractile elements being localized prefereably in the cytoplasm of the longitudinal cell walls.

Actin Polymerization as an Essential Process in Light- and Dark-controlled Chloroplast Anchorage

Summary The effect of the actin-inhibitor cytochalasin B on low fluence-rate chloroplast movements and on Pfr mediated increase of chloroplast anchorage to the cytoplasmic parietal coating of a Mougeotia cell was investigated. We found that cytochalasin B decreases chloroplast anchorage in far-red irradiated and also in darksensitized cells. The results show that actin polymerization is not only involved in light-oriented chloroplast movements but also in the process of chloroplast anchorage to the cytoplasmic parietal coating of the longitudinal cell walls. The results provide a further strong argument for the assumption that both responses, the Pfr-controlled chloroplast movement as well as the Pfr-mediated increase of chloroplast anchorage are realized via similar transduction chains.

ON THE POLARITY OF PHYTOCHROME CONTROLLED LEAF UNFOLDING IN DARK‐GROWN SEEDLINGS OF TRITICUM AESTIVUM*

Abstract— The apical ends of sections cut from etiolated primary wheat leaves show a higher degree of phytochrome‐mediated leaf unfolding than the basal ends, regardless of the position of the segment in the leaf. It could be clarified that this polarity is not the result of a stable gradient within the intact dark grown leaf. Partial irradiation experiments have shown that leaf unfolding can be induced much more effectively by an irradiation of the apical half of a section than of the basal half. Though only the basal end of the sections was incubated with gibberellic acid (GA3) in darkness, the degree of the GA3‐induced leaf unfolding was highest at the apical end of these non‐irradiated segments. The results suggest that in each separate leaf section a new gradient of a still unknown “unfolding‐factor” is established.

THE STIMULATING EFFECT OF A COLD, DARK PRETREATMENT ON THE ETIOPLAST/CHLOROPLAST TRANSFORMATION OF ANGIOSPERMS–II. SYNERGISM BETWEEN RED-LIGHT and COLD PRETREATMENT ON THE CO2- and O2-GAS-EXCHANGE OF WHEAT LEAVES

Abstract— Recently we reported on the stimulating effect of a cold, dark pretreatment on the processes of greening of dark‐grown angiosperms under continuous white light (Schönbohm et al., 1988; Physiol. Plant.72,541–546). These effects could be nullified by a subsequent second dark phase (25°C) which precedes the white light period.

Zur Problematik des In-vivo-Einsatzes von Kalium-Jodid und Natrium-Azid als spezifische Triplett-Quencher von Flavinen

Summary The effect of the flavin-quenchers KI and NaN3 on the red light- and blue light-controlled chloro-plast movements has been investigated. The following results were obtained: 1. Blue light- as well as red light-controlled chloroplast movements are strongly inhibited by KI and by NaN3, respectively. 2. In all types of chloroplast movements, which have been investigated in Mougeotia (IWLM, CWLM, SLM), the inhibitory effect of KI is weakened when exogeneous ATP (or ADP) but not at all ITP (or IDP) is applied simultaneously with KI. 3. Both photosynthetic oxygen evolution and respiration in darkness get inhibited by KI and by NaN3, respectively. 4. KI dissolved in aqua dest. or in sorbitol (10−4 M) almost completely loses its severe inhibitory effect on chloroplast movements. 5. If KI is dissolved in aqua dest. or in sorbitol containing CaCl2 (10−2 M), its strong inhibitory effect upon any light-oriented chloroplast movement is restored. If KI is dissolved together with MgCl2, it restrains chloroplast movement in the weak but not in the strong light. 6. Some experiments demonstrate that the receptibility of the cell for KI is increased by additionally CaCl2. The amount of KI penetrating the cell depends highly on qualities and quantities of light.