Franz Grolig

New trends in photobiology: Light-oriented chloroplast positioning. Contribution to progress in photobiology

Abstract Intracellular rearrangements of chloroplasts with respect to the direction of light, e.g. as found in Vaucheria, Adiantum, Mougeotia and Mesotaenium , are model systems for investigating the processing of light signals from perception through transduction to the terminal response. Two photosensory pigments, namely blue-/UV-absorbing pigment and — in a few exceptional species — the red/far-red reversible phytochrome system, are localized in the cytoplasm outside the chloroplasts. Dichroic orientation of the pigments is important for the detection of light direction via generation of an intracellular absorption gradient. Chloroplast movement, resulting in altered positioning, is based on the actomyosin system. Light-governed reorganization of the cortical and/or subcortical actin cytoskeleton results in biased chloroplast translocation. Besides polymerization or anchoring of actin to cell structures, the activity of myosin may be a point of control in some species. Local changes in membrane properties, including transient modulation of the membrane potential, appear to be the earliest steps in signal tranduction. The role of calcium and calmodulin, obviously involved in some species, is not clear as yet.

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

Summary. We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019–1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.

Differential Ammonia-Elicited Changes of Cytosolic pH in Root Hair Cells of Rice and Maize as Monitored by 2[prime],7[prime]-bis-(2-Carboxyethyl)-5 (and -6)-Carboxyfluorescein-Fluorescence Ratio

Intact hair cells of young rice (Oryza sativa L.) and maize roots (Zea mays L.), grown without external nitrogen, were specifically loaded with 2[prime],7[prime]-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein acetoxymethyl ester to monitor fluorescence ratio cytosolic pH changes in response to external ammonia (NH4+/NH3) application. In neutral media, cytosolic pH of root hairs was 7.15 [plus or minus] 0.13 (O. sativa) and 7.08 [plus or minus] 0.11 (Z. mays). Application of 2 mM ammonia at external pH 7.0 caused a transient cytosolic alkalization (7.5 [plus or minus] 0.15 in rice; 7.23 [plus or minus] 0.13 in maize). Alkalization increased with an increase of external pH; no pH changes occurred at external pH 5.0. The influx of 13N-labeled ammonia in both plant species did not differ between external pH 5.0 and 7.0 but increased significantly with higher pH. Pretreatment with 1 mM 1-methionine sulfoximine significantly reduced the ammonia-elicited pH increase in rice but not in maize. Application of 2 mM methylammonia only caused a cytosolic pH increase at high external pH; the increase in both species compared with the ammonia-elicited alkalization in 1-methionine sulfoximine-treated roots. The differential effects indicate that cytosolic alkalization derived from (a) NH3 protonation after passive permeation of the plasma membrane and, particularly in rice, (b) additional proton consumption via the glutamine synthetase/glutamate synthase cycle.

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.

Effects of NH(4)(+), NO(3)(-) and HCO(3)(-) on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

Abstract. A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pKa = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm2), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH4+ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO3− was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO3−, NO3− elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H+-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO3− provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging.

Gravitropism in Phycomyces: a role for sedimenting protein crystals and floating lipid globules

Abstract. To elucidate the graviperception of the unicellular fungus, Phycomycesblakesleeanus, sporangiophores were inspected for intracellular structures which relocate with respect to gravity. Two structures, paracrystalline proteins (so-called octahedral crystals) and an aggregate of lipid globules, were identified which showed redistribution upon reorientation of the sporangiophore. Octahedral crystals occur throughout the sporangiophore, including the apical growing zone, and are localized inside vacuoles in which they reside singly or in clusters of up to 40 loosely associated individuals. Upon a 90° reorientation of sporangiophores, crystal clusters sedimented in approximately 50–200 s from the upper to the lower side, corresponding to a speed of 0.5–2 μm s−1. Stage-4 sporangiophores (with sporangium) of three mutants which lack the crystals displayed anormal kinetics of gravitropism and substantially reduced bending angles in comparison to sporangiophores of the wild type. While horizontally placed wild-type sporangiophores reached the vertical position after 10–12 h, the crystal-lacking mutants bent maximally 40°–50° upward. In stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 μm below the apex. The globules floated upwards when the sporangiophore was placed horizontally forming in this way a cap-like aggregate. It is proposed that both the sedimenting protein crystals and the upward-floating globules are involved in gravisensing.

Myosin heavy chains: Detection by immuno-blotting in higher plants and localization by immunofluorescence in the alga chara

Abstract A monoclonal antibody to the heavy chain of myosin from mouse 3T3 cells was used to identify myosin heavy chains in four flowering plants and to identify and localize them in the green alga Chara. The Mr of the immunoreactive bands varied from ca 200 000 in Chara and Arabidopsis to 170 000 in mung beans, peas and wheat. An additional band of 158 000-Mr was resolved in roots and shoots of mung beans. Chara contained a second, immunoreactive band of 110 000-Mr whose possible relationship to the tail-less myosin I enzymes is discussed. Immunofluorescence of giant internodal cells of Chara showed that myosin was almost entirely confined to the streaming endoplasm. Individual organelles and beaded endoplasmic strands were heavily labelled as were the sub-cortical filament bundles. Actin, in contrast, was confined to the sub-cortical bundles. It is proposed that force is generated by interaction of the actin in the subcortical bundles with myosin on individual organelles and on the beaded endoplasmic strands. By ramifying through the endoplasm, the strands may ensure the cohesive movement of the whole mass of endoplasm.

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris, and their relevance to chloroplast movement.

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pKa-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.109 dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

Abstract. The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold.

Properties of monoclonal antibodies to plant calmodulin

Abstract Ca2+-binding proteins link changes in cytoplasmic free Ca2+ concentration to physiological effects. Two monoclonal antibodies raised against purified calmodulin, the best documented such protein in plants, bound plant calmodulin in enzyme-linked immunoassays and immunoprecipitation. Only one antibody recognised sodium dodecyl sulphate-denatured plant calmodulin while mammalian calmodulin was recognised with substantially lower efficiency. Immunoblotting crude extracts from a variety of angiosperms revealed two bands of similar Mr that showed Ca2+-dependent mobility shifts on sodium dodecyl sulphate gel electrophoresis. Carboxypeptidase B digestion increased the proportion of the slowly moving band consistent with it being calmodulin from which the C-terminal lysine residue had been removed. The reaction may occur in vivo since both forms, but not a presumed proteolytic fragment of lower Mr are detected when tissues are extracted directly into trichloroacetic acid to inhibit post-homogenisation proteolysis.