Secretion of Lectin-Binding Material in Rhizoid Differentiation of Spirogyra

Some species of Spirogyra (a green alga) anchor to the substratumby differentiating the rhizoid. The transparent material secretedat the tip of the terminal cell or rhizoid is speculated tobe glycoprotein (Nagata 1977). To examine this, we treated Spirogyrafilaments with differentiating rhizoid with fluorescently labeledlectins. Among the nineteen lectins examined, only Bandeiraea(Griffonia) simplicifolia lectin I (BSL-I) strongly stainedthe transparent material, suggesting that the transparent materialcontains     

Rhizoid differentiation in Spirogyra: position sensing by terminal cells

Some species of Spirogyra anchor themselves to the substrate by differentiating rhizoids. A rhizoid is differentiated only from the terminal cell, suggesting that this cell can recognize its terminal position in a filament. In the present study, we have analyzed the mechanism for position sensing by the terminal cell. When a filament is cut, a new cell occupies the terminal position, and three phenomena are induced: (1) the cell wall of the cut cell detaches from the new terminal cell; (2) adhesive material is secreted by the terminal cell; and (3) the terminal cell begins to differentiate a rhizoid via tip growth. All of these phenomena were inhibited by adding sorbitol to the external medium, suggesting that turgor pressure is involved in position sensing by the terminal cell. The inhibition by sorbitol was reversible. Upon cutting a filament, the distal end of a new terminal cell became convex. However, when a filament was cut in the presence of sorbitol, the distal end of a new terminal cell became less convex. Either treatment with Gd(3+) or decrease in extracellular Ca(2+) resulted in inhibition of all these phenomena, suggesting possible involvement of stretch-activated ion channel in position sensing by terminal cells.     

Rhizoid differentiation in Spirogyra I. Basic features of rhizoid formation

Several types of rhizoids occurring in the process of differentiationin Spirogyra sp. are described and their interrelation was elucidated.There are two differentiation sequences: P_pPRh_ros or PpPRh_rodRh_ros(for explanation of abbreviations see p. 533), although undersome conditions the sequences ceased halfway through. The initiationtime for rhizoid formation had no relation to the cell cyclestage. The difference in growth patterns between the rhizoidand ordinary filament cells was demonstrated with Calcofluor-stainingand centrifugation. The optimum temperature and pH of the culture medium for rhizoiddifferentiation were 20?C and pH 7, respectively. A contactstimulus was not necessary for induction. Of the several environmental factors examined, light was themost important, for rhizoid formation, since a rhizoid was inducedonly when light was given after cutting the filament. (Received December 14, 1972; )     

Inhibition of Etiolation in Spirogyra by Phytochrome

Cells of Spirogyra filaments grown in darkness become longer than light grown cells. This elongation can be prevented by a few minutes of red light given with intervals of 24 h under the dark period. Far-red light given after the red light pulse counteracts the red light effect. Cells which have finished their elongation in light do not elongate further in darkness. Along with the cell elongation the chloroplasts become less spirally wound and ultimately shortened to straight bands.     

Rhizoid differentiation in Spirogyra II. Photoreversibility of rhizoid induction by red and far-red light

The optimum light conditions for rhizoid formation in Spirogyrawere determined. Red light was significantly effective on rhizoidformation while green, blue and violet lights had less effect.The dose-effect curve of red light was investigated and theminimum energy needed to saturate the effect was 8.1 Kergs.cm_–2.The effect of red light was completely reversed by subsequentirradiation with far-red light. The doseeffect curve of far-redlight was also obtained. The repeatedly reversible photoresponseswith red and far-red light strongly suggest that the photoreceptorof the rhizoid formation system is phytochrome. The existenceof phytochrome in the Spirogyra cell was also demonstrated spectrophotometrically.The half time for the escape reaction from the reversal effectof far-red light was 2hr. There may be no pigment other thanphytochrome mediating the photoreaction. (Received December 14, 1972; )     

Intracellular Localization and Dichroic Orientation of Phytochrome in Plasma Membrane and/or Ectoplasm of a Centrifuged Protonema of Fern Adiantum capillus-veneris L.

Protonemata of the fern Adiantum capillus-veneris grown undercontinuous red light for 6 days were kept in darkness for 15h and subsequently centrifuged 3 times in different directions,so that oil droplets and other cytoplasm were removed from theapical region of the protonemata. Electron micrographs clearlydemonstrated that cell wall, plasma membrane, ectoplasm andmicrotubules remained in the apical and subapical regions afterthe centrifugal treatments. A brief local exposure of the flankof the subapical region of the centrifuged protonemata to amicrobeam of red light effectively induced a phototropic responsetoward the irradiated side, suggesting that phytochrome is locatedin the ectoplasm and/or plasma membrane. When the flank of thecentrifuged protonema was irradiated with linearly polarizedred or far-red light, red light with an electrical vector parallelto the cell surface was more effective than that perpendicularto the cell surface. The direction of the electrical vectorof far-red light for reversion of the preirradiated red lighteffect, however, was opposite. These results suggest that differentdichroic orientations of P_R and P_FR exist in the plasma membraneor ectoplasm. (Received May 26, 1983; Accepted September 1, 1983)     

Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Intracellular Localization of Nitrate Reductase in Neurospora crassa

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.     

Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves

The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.     

Phytochrome evolution: Phytochrome genes in ferns and mosses

We have isolated phytochrome genes from the moss Physcomitrella , the fern Psilotum and PCR-generated phytochrome sequences from a few other ferns. The phytochrome gene of the moss Physcomitrella turned out not to contain the aberrant C-terminal third of the phytochrome from the moss Ceratodon , but the transmitter module-like sequences found in other phytochromes. A series of different phytochrome genes was detected in Psilotum . Differences between the amino acid sequences derived from them ranged from about 5 to more than 22%. Some of these genes are likely pseudogenes. Analysis by phylogenetic tree constructions revealed that higher and lower plant phytochromes evolved with different velocities. Lower plant phytochromes form a separate family characterized by a high degree of similarity. The amino acid differences between phytochrome types detected in a single species of higher plants are about two-fold higher than the differences between phytochromes of species of lower plants belonging to different divisions ( Physcomitrella and Selaginella ). Future studies on phytochrome sequences may eventually also throw light on the significance of Psilotum in the evolution of vascular plants.     

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in (p)O(2), and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in (p)O(2) during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high (p)O(2). E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O(2) (-) which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O(2) (-). On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O(2) (-), whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O(2) (-).     

The Cytological Localization of Intracellular Neuronal Acetylcholinesterase

Sections of cat ciliary ganglia were stained for acetylcholinesterase activity by several modifications of the acetylthiocholine method in order to achieve optimal accuracy of cytological localization of the enzyme. These were compared by ordinary light and phase contrast microscopy with similar sections stained by standard techniques for Nissl substance, the Golgi apparatus, and the neurofibrillae, and by intravital methylene blue. The pattern of cytoplasmic distribution of acetylcholinesterase corresponded most closely with that of the Nissl substance. Following total inactivation of the ganglionic acetylcholinesterase by intravenously administered di-isopropyl fluorophosphate, the reappearance of the enzyme in vivo occurred at the same cytoplasmic sites prior to its reappearance at the cell membrane or preganglionic axonal terminations. These observations, and reports cited from the literature, provide support for the hypothesis that acetylcholinesterase is synthesized within the endoplasmic reticulum, then transported via its canaliculi to the surface of the cell and its processes, where its functional sites are oriented externally to the lipoidal membrane.     

27. Gerhard Reichart: Die Synchronkultur von Spirogyra

Es wurden Bedingungen ermittelt, welche eine synchrone Kultur und vegetative Vermehrung von Spirogyra majuscula erlauben. Herrn Prof. Dr. W. Baumeister danke ich herzlich für personelle und apparative Unterstützung dieser Arbeit. Fräulein B. Zielstorff danke ich für ihre zuverlässige technische Mitarbeit.     

Differentiation in Coprinus lagopus. III. Expansion of excised fruit-bodies.

Fruit-body expansion was studied in Coprinus lagopus (sensu Buller) following surgical procedures. Elongation occurred after denuding mushroom caps of essentially all peripheral scales. Young primordia (1 - 5 mm) failed to develop after vertical bisection. Older primordia (e.g. 10 mm) expanded 3 - 4 fold after vertical bisection or quadrisection, underwent autolysis and basidiospore production. An amorphous brown gel in the stripe lumen disappeared during development of bisected primordia. Stripes isolated from primordia expanded autonomously and exhibited negative geotropism when incubated upside down or when the stripe apex was removed. Displacement of charcoal particles dusted on intact stripes revealed the most active zone of expansion to be the upper mid-region of the stripe. Segmented stripes likewise showed most active elongation in the mid-region. Vertically bisected stripes also expanded.     

Negative Phototropism in the Rhizoid of Bryopsis plumosa

Negative phototropism in the rhizoid of Bryopsis plumosa wasanalyzed. The growth zone of the rhizoid was confined to theapical hemisphere, as is typical of tip growth. Upon unilateralillumination, the rhizoid bent away from the light source witha "bulging" manner. The photoreceptive site for phototropismwas also restricted to the apical hemisphere. The action spectrumfor this negative phototropism was determined from fluence-responsecurves that were obtained after fixing the duration of illuminationat 60 min and varying the fluence rate between 0.1 and 3.0 Wm^-2. The action spectrum had a large peak at 467 nm and smallerpeaks at 378 and 414 nm, resembling the action spectra of certainother blue-light responses. (Received November 29, 1995; Accepted May 29, 1995)