Gottfried Wagner

Bioenergetic role of halorhodopsin in Halobacterium halobium cells

Bioenergetic functions in the membranes of Halobacterium halobium depend on respiration in the dark, and on two retinal pigments during illumination. The better known of these is bacteriorhodopsin [ l ], which is found in crystalline arrays (patches) covering as much as 50% of the cell surface. Bacteriorhodopsin is a light-dependent pump for protons, and it has been proposed [2-4] that the pH difference and particularly the electrical potential, which is created during illumination by proton extrusion across the cytoplasmic membrane, is the energizing folce for ATP synthesis. Illumination will indeed support the growth of bacteriorhodopsin-containing H. halobium under strictly anaerobic conditions [5]. The second retinal pigment, present in smaller amounts in the halobacteria, is halorhodopsin. This pigment is a light-driven electrogenic pump for Na ÷ [6-8], and its spectral characteristics are somewhat different [9,10] from those of bacteriorhodopsin. The presence of this pump is most easily recognized in cell envelope vesicles prepared from H. halobium strains lacking bacteriorhodopsin. Illumination of such vesicles results in uncoupler-facilitated passive proton uptake until a pH difference (acid inside) is balanced by a membrane potential (negative inside), so that the protonmotive force is zero [8]. Appropriately prepared vesicles from wild-type, bacteriorhodopsin-containing cells show both active and passive proton movements upon illumination [7,8], the former appearing as efflux, the latter as influx. Proton conductors will tend to suppress proton efflux but enhance the influx. It appears therefore, that when present, both pumps are oriented in the outward direction in

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca2+-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca2+-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC50) of chloroplast movement is caused by 20–50 μmol l-1 trifluoperazine or 100 μmol l-1 W-7. The Ca2+-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.

DIFFERENTIAL EFFECT OF CALCIUM ON CHLOROPLAST MOVEMENT IN MOUGEOTIA

The flat, ribbon‐shaped chloroplast in the filamentous green alga Mougeotia sp. undergoes light‐induced orientational movement controlled by an intracellular tetrapolar gradient of the active form of phytochrome, Pfr. Some substructural and physiological aspects of this reaction were studied. An intracellular pattern of microfilaments (diameter: 5–10nm), presumably related to chloroplast movement, was identified in situ. In addition, it could be shown that chloroplast movement decreases parallel to a nitric acid soluble fraction of intracellular calcium. These results might indicate that phytochrome governs chloroplast movement in Mougeotia via control of the binding state of calcium.

The role(s) of calcium ions in phytochrome action.

Phytochrome, the red (R)*and far-red light (FR)absorbing morphogenetic photoreceptor, which occurs throughout the Plant Kingdom, was discovered by scientists at the US Department of Agriculture, Beltsville, MD. This year is the 40th anniversary of the prediction of the R/FR reversible pigment in plants and more than 30 years since its first spectroscopic detection. During the last four decades extensive progress has been made in understanding the molecular structure and function of phytochrome (for review see Furuya, 1987). Phytochrome genes have now been cloned for a few plant species (Quail et al . , 1987; Furuya, 1989; Sharrock and Quail, 1989). The photoregulation of genes, including the phytochrome gene itself, has been extensively studied (Quail et a / . , 1987; Nagy et a / . , 1988; Furuya, 1989; Tomizawa et a[., 1990). However, the molecular mechanism of phytochrome action is still obscure. Multiple response types could be an indication of different modes of action of the photoreceptor (Jordan et al., 1986; Kronenberg and Kendrick. 1986; Schafer et al., 1986; Furuya, 1989). One attractive hypothesis is that calcium ions (Ca2+) participate as a second messenger (Roux, 1984). During the last decade many papers have been published about the involvement of Ca” in the regulation of different phytochrome-regulated processes. Some o’f them were reviewed by Roux et 121. in 1986. Since that time

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)-buffered media (-log [Ca2+] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca2+]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca2+] in M. scalaris that was nearly independent of the external [Ca2+] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca2+ from intracellular stores. Increased cytoplasmic [Ca2+] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca2+-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.

Mechanism of chloroplast movement inMougeotia

SummaryThe single, ribbon-shaped chloroplast in the filamentous green algaMougeotia performs orientational movements with respect to light. The chain of reaction involves phytochrome as the photoreceptor pigment to perceive the light signal differentiated by wavelength and direction, calcium probably to convert the light signal into a chemical message and actomyosin to respond to this message and to move the chloroplast accordingly.Precise reorientation of the chloroplast is proposed to be brought about by a dual function of phytochrome: regulation of the cellular level of calciumand regulation of membrane anchorage sites to actin.

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia.

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.

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

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 μM, 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.

Heavy-meromyosin-decoration of microfilaments from Mougeotia protoplasts

The cell wall of the green alga Mougeotia was enzymatically digested by macerase and cellulysin. Released protoplasts were spread on poly-L-ornithine, formvar-carbon-coated grids, and cell fragments were collected for structural characterization. Large numbers of 5–7 nm filaments are seen which may be decorated with heavy meromyosin (HMM), a digest product of muscle myosin that binds specifically to actin, supporting the hypothesis that the phytochrome-mediated chloroplast movements in these algae are driven by a contractile complex of actomyosin.

PHYTOCHROME OF THE GREEN ALGA MOUGEOTIA : CDNA SEQUENCE, AUTOREGULATION, AND PHYLOGENETIC POSITION

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strain-specific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5′ non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52–53% for PHYA and 57–59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under Pr conditions, phytochrome mRNA levels were tenfold higher than under Pfr conditions.