Randy Wayne

Gravity-dependent polarity of cytoplasmic streaming in Nitellopsis

SummaryThe internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K+ channels. The K+ channel blocker TEA Cl− inhibits the NR effect.External Ca2+ is required for normal graviresponsivness. The [Ca2+] of the medium determines the polarity of cytoplasmic streaming. Less than 1 μM Ca2+ resulted in a PR<1 while greater than 1 μM Ca2+ resulted in the normal gravity response. The voltage-dependent Ca2+ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl3 resulted in a PR<1, indicating the presence of two types of Ca2+ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.

The excitability of plant cells: with a special emphasis on characean internodal cells.

This review describes the basic principles of electrophysiology using the generation of an action potential in characean internodal cells as a pedagogical tool. Electrophysiology has proven to be a powerful tool in understanding animal physiology and development, yet it has been virtually neglected in the study of plant physiology and development. This review is, in essence, a written account of my personal journey over the past five years to understand the basic principles of electrophysiology so that I can apply them to the study of plant physiology and development.My formal background is in classical botany and cell biology. I have learned electrophysiology by reading many books on physics written for the lay person and by talking informally with many patient biophysicists. I have written this review for the botanist who is unfamiliar with the basics of membrane biology but would like to know that she or he can become familiar with the latest information without much effort. I also wrote it for the neurophysiologist who is proficient in membrane biology but knows little about plant biology (but may want to teach one lecture on “plant action potentials”). And lastly, I wrote this for people interested in the history of science and how the studies of electrical and chemical communication in physiology and development progressed in the botanical and zoological disciplines.ÜbersichtDieser Überblick beschreibt die Grundprinzipen der Electrophysiologie unter Verwendung eines Aktionspotentials in internodalen Zellen der characean Algen als pädagogeshes Mittel. Die Elektrophysiologie hat sich beim Verständnis von Tierphysiologie und entwicklung als wirksames Mittel erwiesen; dennoch ist sie bisher beim Studium der Pflanzenphysiologie und entwicklung praktish vernachlässigt worden. Dieser Überblick ist im Wesentlichen ein schriftlichen Bericht meiner persönlichen Bemühungen in den letzten fünf Jahren die Grungprinzipen der Electrophysiologie zu verstehen, um sie auf das Studium der Pflanzenphysiologie und entwicklung anzuwenden.Meine Spezialität ist klassische Botanik und Zellbiologie. Elektrophysiologie habe ich durch das Lesen von vielen Physikbüchern für den Laien erlerut; daneben hatte ich auch die Gelengheit, informell mit vielen geduldigen Biophysikern darüber zu sprechen. Ich habe diesen Überblick für den Botaniker geschreiben, der mit den Grundlagen der Membranbiologie nicht vertraut ist, der aber wissen möchte, dass er/sie ohne allerzu grossen Aufwand mit dem neuesten Stand der Wissenschaft vertraut werden kann. Ich habe ihn weiterhin für den Neurophysiologen geschrieben, der mit der Membranphysiologie wohl vertraut ist, der aber wenig über Pflanzenbiologie weiss (aber velleicht eine Vorlesung über “Pflanzenaktionspotentials” halten möchte). Schlieslich habe ich ihn auch für den Leser geschrieben, der sich für die Wissenschaftsgeschichte interessiert und dafür, wie das Studium der Elektrischen und chemischen Kommunikation in der Physiologie und Entwicklung in den botanischen und zoologischen Disziplinen fortgeschritten ist.ResumenEste estudio analiza los principios básicos de electrofisiología utilizando la generatión de un potential de actión en células internodiales de algas characean como herramienta pedagógica. Electrofisiología ha demostrado ser un excelente método para el entendimiento de la fisiología y del desarrollo animal pero ha sido ignorada en el estudio de la fisiología y del desarrollo de plantas. Este estudio es, esencialmente, un testimonio de mi experiencia personal durante los últimos cinco años en los que he estado estudiando los principios básicos de electrofisiología para poder aplicarlos al estudio de fisiología y desarrollo de plantas.Mis estudios se concentraron en botánica clásica y biología celular. He aprendido electrofisiología a base de leer muchos libros de física escritos para gente sin gran conocimiento de la materia, además he tenido el placer de hablar informalmente con muchos biofísicos de gran paciencia. He escrito este estudio para el botánico que desconoce la base de biologfa membranal pero al que le gustaría saber que él o ella puede familiarizarse con la informatión más actual sin mucho esfuerzo. También lo escribí para el neurofisiólogo, experto en biología membranal, pero que no sabe mucho sobre biología de plantas (y que desee enseñar una clase sobre los “potenciales de actión de las plantas”). Y finalmente, escri’i esto para gente interesada en la historia de la ciencia y en cómo los estudios de comunicación eléctrica y química en la fisiología y el desarrollo progresaron en las disciplinas zoolígicas y botánicas.

Hydrostatic pressure mimics gravitational pressure in characean cells

SummaryHydrostatic pressure applied to one end of a horizontalChara cell induces a polarity of cytoplasmic streaming, thus mimicking the effect of gravity. A positive hydrostatic pressure induces a more rapid streaming away from the applied pressure and a slower streaming toward the applied pressure. In contrast, a negative pressure induces a more rapid streaming toward and a slower streaming away from the applied pressure. Both the hydrostatic pressure-induced and gravity-induced polarity of cytoplasmic streaming respond identically to cell ligation, UV microbeam irradiation, external Ca2+ concentrations, osmotic pressure, neutral red, TEA Cl−, and the Ca2+ channel blockers nifedipine and LaCl3. In addition, hydrostatic pressure applied to the bottom of a vertically-oriented cell can abolish and even reverse the gravity-induced polarity of cytoplasmic streaming. These data indicate that both gravity and hydrostatic pressure act at the same point of the signal transduction chain leading to the induction of a polarity of cytoplasmic streaming and support the hypothesis that characean cells respond to gravity by sensing a gravity-induced pressure differential between the cell ends.

Purification and characterization of sorbitol-6-phosphate phosphatase from apple leaves

Abstract Sorbitol-6-phosphate phosphatase (SorPP; EC 3.1.3.50) catalyzes the final step in sorbitol biosynthesis in sorbitol-synthesizing plant species, but its kinetic and regulatory properties have not been characterized. In this study, the enzyme was purified 1727-fold to apparent homogeneity from apple leaves with a maximal specific activity of 89.8 μmol min−1 mg−1 protein measured at 2 mM sorbitol-6-phosphate (sorbitol-6-P). The enzyme is a monomer with a molecular mass of 61 kDa. The enzyme is highly specific for sorbitol-6-P with a Km of 0.85 mM and is unable to cleave other phosphate esters at a significant rate. The activity is absolutely dependent on Mg2+ with a Km of 0.29 mM at an optimal pH of 6.8. Fluoride, vanadate, molybdate, and inorganic phosphate inhibit SorPP activity. Sorbitol is a competitive inhibitor for SorPP with a Ki of 109 mM. The possible feedback mechanism for the regulation of sorbitol biosynthesis is also discussed.

Calcium-regulated appressorium formation of the entomopathogenic fungusZoophthora radicans

SummaryThe fungusZoophthora radicans (Zygomycetes: Entomophthorales) requires external Ca2+ for appressorium formation but not for conidial germination. The number of appressoria formed depends on the Ca2+ concentration of the medium. At low [Ca2+] (100 pM) nuclear division and germ tube growth are significantly reduced compared to higher Ca2+ concentrations (10 and 1,000 μM). By contrast, neither external K+ nor external Cl− is needed for germination or appressorium formation. Treatment of conidia with a Ca2+-antagonist, Nd3+, and a Ca2+-channel blocker, nifedipine, inhibits appressorium formation, showing that a Ca2+ influx is required for appressorium formation. Furthermore, the partial yet saturating inhibition by nifedipine and complete inhibition by Nd3+ indicates that at least two kinds of Ca2+ channels are involved in appressorium formation. A contribution of intracellular Ca2+ to the signal transduction chain for the formation of appressoria is demonstrated by the inhibitory effect of the intracellular Ca2+ antagonist TMB-8. The calmodulin antagonists R24571, TFP, W-7, and W-5 inhibit appressorium formation at concentrations which have no effect on germination. The data presented in this paper are consistent with the hypothesis that a Ca2+/calmodulin system is involved in regulating appressorium formation. However, since the direct effects of the drugs were not specifically tested on their proposed binding sites, we leave room for alternative hypotheses that have yet to be formulated.

Detection of gravity-induced polarity of cytoplasmic streaming in Chara

SummaryGravity induces a polarity of cytoplasmic streaming in vertically-oriented internodal cells of characean algae. The motive force that powers cytoplasmic streaming is generated at the ectoplasmic/endoplasmic interface. The velocity of streaming, which is about 100 μm/s at this interface, decreases with distance from the interface on either side of the cell to 0 μm/s near the middle. Therefore, when discussing streaming velocity it is necessary to specify the tangential plane through the cell in which streaming is being measured. This is easily done with a moderate resolution light microscope (which has a lateral resolution of 0.6 μm and a depth of field of 1.4 μm), but is obscured when using any low resolution technique, such as low magnification light microscopy or laser Doppler spectroscopy. In addition, the effect of gravity on the polarity of cytoplasmic streaming declines with increasing physiological age of isolated cells. Using a classical mechanical analysis, we show that the effect of gravity on the polarity of cytoplasmic streaming cannot result from the effect of gravity acting directly on individual cytoplasmic particles. We suggest that gravity may best be perceived by the entire cell at the plasma membrane-extracellular matrix junction.

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.

Fibre cables in the lacunae of Typha leaves contribute to a tensegrity structure

BACKGROUND AND AIMS Cables composed of long, non-lignified fibre cells enclosed in a cover of much shorter thin-walled, crystal-containing cells traverse the air chambers (lacunae) in leaves of the taller species of Typha. The non-lignified fibre cables are anchored in diaphragms composed of stellate cells of aerenchyma tissue that segment the long air chambers into smaller compartments. Although the fibre cables are easily observed and can be pulled free from the porous-to-air diaphragms, their structure and function have been ignored or misinterpreted. METHODS Leaves of various species of Typha were dissected and fibre cables were pulled free and observed with a microscope using bright-field and polarizing optics. Maximal tensile strength of freshly removed cables was measured by hanging weights from fibre cables, and Instron analysis was used to produce curves of load versus extension until cables broke. KEY RESULTS AND CONCLUSIONS Polarized light microscopy revealed that the cellulose microfibrils that make up the walls of the cable fibres are oriented parallel to the long axis of the fibres. This orientation ensures that the fibre cables are mechanically stiff and strong under tension. Accordingly, the measured stiffness and tensile strength of the fibre cables were in the gigapascal range. In combination with the dorsal and ventral leaf surfaces and partitions that contain lignified fibre bundles and vascular strands that are strong in compression, the very fine fibre cables that are strong under tension form a tensegrity structure. The tensegrity structure creates multiple load paths through which stresses are redistributed throughout the 1-3 m tall upright leaves of Typha angustifolia, T. latifolia, T. × glauca, T. domingensis and T. shuttleworthii. The length of the fibre cables relative to the length of the leaf blades is reduced in the last-formed leaves of flowering individuals. Fibre cables are absent in the shorter leaves of Typha minima and, if present, only extend for a few centimetres from the sheath into the leaf blade of Typha laxmannii. The advantage of the structure of the Typha leaf blade, which enables stiffness to give way to flexibility under windy conditions, is discussed for both vegetative and flowering plants.

Ultrarapid endocytotic uptake of large molecules inDunaliella species

SummaryThis paper describes the uptake of Lucifer Yellow carbohydrazide and fluorescent dextrans labeled with fluorescein isothiocyanate or Sodium Green (molecular masses ranging from 522 to 2 × 106 Da) byDunaliella spp. halotolerant unicellular green algae isolated from salt pools in the Sinai peninsula. The fluorescent dyes were taken up into a set of vesicles around the nucleus and just above the chloroplast. It proved impossible to inhibit uptake of the fluorescent compounds in cells treated with a large variety of metabolic and other inhibitors. Cell labeling was complete within half a minute of addition of fluorescent compounds to the outside medium; efflux was equally rapid. The results are interpreted in terms of an endocytotic process whereby the outside medium, together with any substance dissolved in it, remains within vesicles enclosed within the cell body but cycles rapidly between the plasma membrane and the interior of the cell. The outside medium does not pass across the vesicular membrane, nor enters the cytosol.

Button botany: plasmodesmata in vegetable ivory

The hard endosperm of species of the palm genus Phytelephas (elephant plant), known as vegetable ivory, was used in the manufacture of buttons in the nineteenth century, the early twentieth century, and again in more recent times. Here, we show that the pathways for intercellular communication, including the cytoplasm in opposite pits and the plasmodesmata that traverse the cell wall, can be visualized in century-old inexpensive buttons that are readily available in antique shops.