Alison R. Taylor

Cell Fate Determination by the Cell Wall in Early Fucus Development

In multicellular plants, development starts with an asymmetric division of the zygote into two differentiated cells. The nature and distribution of fate-determining factors operating during embryogenesis remain largely obscure. Laser microsurgery was used here to dissect two-celled embryos of the alga Fucus spiralis. Removal of protoplasts from the cell wall induced dedifferentiation. However, isolated cells within the walls followed their restricted fate. Moreover, contact of one cell type with the isolated cell wall of the other cell type caused its fate to be switched. The cell wall thus appears to maintain the differentiated state and to direct cell fate in plant development.

Spatiotemporal Patterning of Reactive Oxygen Production and Ca2+ Wave Propagation in Fucus Rhizoid Cells

Both Ca2+ and reactive oxygen species (ROS) play critical signaling roles in plant responses to biotic and abiotic stress. However, the positioning of Ca2+ and ROS (in particular H2O2) after a stress stimulus and their subcellular interactions are poorly understood. Moreover, although information can be encoded in different patterns of cellular Ca2+ signals, little is known about the subcellular spatiotemporal patterns of ROS production or their significance for downstream responses. Here, we show that ROS production in response to hyperosmotic stress in embryonic cells of the alga Fucus serratus consists of two distinct components. The first ROS component coincides closely with the origin of a Ca2+ wave in the peripheral cytosol at the growing cell apex, has an extracellular origin, and is necessary for the Ca2+ wave. Patch-clamp experiments show that a nonselective cation channel is stimulated by H2O2 and may underlie the initial cytosolic Ca2+ increase. Thus, the spatiotemporal pattern of the Ca2+ wave is determined by peripheral ROS production. The second, later ROS component localizes to the mitochondria and is a direct consequence of the Ca2+ wave. The first component, but not the second, is required for short-term adaptation to hyperosmotic stress. Our results highlight the role of ROS in the patterning of a Ca2+ signal in addition to its function in regulating cell wall strength in the Fucus embryo.

A Voltage-Gated H+ Channel Underlying pH Homeostasis in Calcifying Coccolithophores

Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths) in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO3 − as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan Hv1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the Hv1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate use of H+ derived from calcification for intracellular CO2 generation. Furthermore, the presence of Hv1 class ion channels in a wide range of extant eukaryote groups indicates they evolved in an early common ancestor.

Proton channels in algae: reasons to be excited

A fundamental requirement of all eukaryotes is the ability to translocate protons across membranes. This is critical in bioenergetics, for compartmentalized metabolism, and to regulate intracellular pH (pH(i)) within a range that is compatible with cellular metabolism. Plants, animals, and algae utilize specialized transport machinery for membrane energization and pH homeostasis that reflects the prevailing ionic conditions in which they evolved. The recent characterization of H(+)-permeable channels in marine and freshwater algae has led to the discovery of novel functions for these transport proteins in both cellular pH homeostasis and sensory biology. Here we review the potential implications for understanding the origins and evolution of membrane excitability and the phytoplankton-based marine ecosystem responses to ocean acidification.

Calcification and inorganic carbon acquisition in coccolithophores

A number of species of coccolithophorid phytoplankton precipitate calcite inside intracellular vesicles (coccolith vesicles). They can form vast blooms under certain conditions, and account for major fluxes of inorganic carbon (Ci) to the ocean floor. The functions of calcification have been debated for many years, and a role in carbon acquisition has been proposed by several workers. The precipitation of calcite from HCO3- involves the production of protons that can potentially be used to facilitate the use of external HCO3- as a photosynthetic substrate. For this function to be feasible, certain criteria must be met. HCO3- (rather than CO32-) should be the external substrate for calcification, photosynthesis should be facilitated by HCO3- in calcifying cells when CO2 availability is limiting, and the transport of Ci and Ca2+ to the site of calcification should be energetically and kinetically feasible. Considerable evidence exists for HCO3- as the substrate for calcification in coccolithophores. However, evidence for a direct role for calcification in supply of Ci for photosynthesis is less clear. The environmental factors that regulate calcification are still uncertain but appear to be related as much to the availability of nutrients as CO2. Transport of Ci to the intracellular site of calcification and removal of H+ from the coccolith vesicle appear to present few energetic or kinetic constraints. However, the large sustained transcellular fluxes of Ca2+ required for calcification probably occur via a pathway that does not involve diffusion across the cytoplasm.

Laser microsurgery of higher plant cell walls permits patch-clamp access.

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1–3 [mu]m in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K+-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

THE ROLE OF NITRIC OXIDE IN DIATOM ADHESION IN RELATION TO SUBSTRATUM PROPERTIES1

Adhesion of raphid diatoms to surfaces, mediated by the secretion of extracellular polymeric substances (EPS), is an important strategy for growth and survival. Diatom biofilms are also important in the context of biofouling. Diatoms exhibit selectivity in adhering to surfaces, but little is understood about how they perceive the properties of a substratum and translate that perception into altered adhesion properties. In this study, we demonstrate that Seminavis robusta Danielidis et D. G. Mann, like many other pennate diatoms, adheres more strongly to hydrophobic surfaces (such as silicone elastomer foul‐release coatings) than to hydrophilic surfaces. To explore the cellular mechanisms that may underlie this selectivity, we tested the hypothesis that diatoms may perceive a hydrophilic surface as unconducive to adhesion through a form of stress response involving nitric oxide (NO) production. Single‐cell imaging with the fluorescent indicator DAF‐FM DA (4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate), revealed NO levels that were 4‐fold higher in cells adhered to a hydrophilic surface (acid‐washed glass) compared with a hydrophobic surface (polydimethylsiloxane elastomer, PDMSE). Elevated levels of NO caused by the addition of the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) did not affect growth, but cells showed reduced adhesion strength to both glass and PDMSE. Addition of the nitric oxide synthase inhibitor NG‐monomethyl‐l‐arginine (NMMA) caused a small but significant increase in adhesion strength. Overall, the results suggest that NO acts as a signal of the wettability properties of substrata for Seminavis.

A role for diatom-like silicon transporters in calcifying coccolithophores

Biomineralization by marine phytoplankton, such as the silicifying diatoms and calcifying coccolithophores, plays an important role in carbon and nutrient cycling in the oceans. Silicification and calcification are distinct cellular processes with no known common mechanisms. It is thought that coccolithophores are able to outcompete diatoms in Si-depleted waters, which can contribute to the formation of coccolithophore blooms. Here we show that an expanded family of diatom-like silicon transporters (SITs) are present in both silicifying and calcifying haptophyte phytoplankton, including some globally important coccolithophores. Si is required for calcification in these coccolithophores, indicating that Si uptake contributes to the very different forms of biomineralization in diatoms and coccolithophores. Significantly, SITs and the requirement for Si are absent from highly abundant bloom-forming coccolithophores, such as Emiliania huxleyi. These very different requirements for Si in coccolithophores are likely to have major influence on their competitive interactions with diatoms and other siliceous phytoplankton.

A novel Cl- inward-rectifying current in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus.

We investigated the membrane properties and dominant ionic conductances in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus using the patch-clamp technique. Whole-cell recordings obtained from decalcified cells revealed a dominant anion conductance in response to membrane hyperpolarization. Ion substitution showed that the anion channels were selective for Cl− and Br− over other anions, and the sensitivity to the stilbene derivative 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, ethacrynic acid, and Zn2+ revealed a pharmacological profile typical of many plant and animal anion channels. Voltage activation and kinetic characteristics of the C. pelagicusCl− channel are consistent with a novel function in plants as the inward rectifier that tightly regulates membrane potential. Membrane depolarization gave rise to nonselective cation currents and in some cases evoked action potential currents. We propose that these major ion conductances play an essential role in membrane voltage regulation that relates to the unique transport physiology of these calcifying phytoplankton.

A Fast Na+/Ca2+-Based Action Potential in a Marine Diatom

Background Electrical impulses in animals play essential roles in co-ordinating an array of physiological functions including movement, secretion, environmental sensing and development. Underpinning many of these electrical signals is a fast Na+-based action potential that has been fully characterised only in cells associated with the neuromuscular systems of multicellular animals. Such rapid action potentials are thought to have evolved with the first metazoans, with cnidarians being the earliest representatives. The present study demonstrates that a unicellular protist, the marine diatom Odontella sinensis, can also generate a fast Na+/Ca2+ based action potential that has remarkably similar biophysical and pharmacological properties to invertebrates and vertebrate cardiac and skeletal muscle cells. Methodology/Principal Findings The kinetic, ionic and pharmacological properties of the rapid diatom action potential were examined using single electrode current and voltage clamp techniques. Overall, the characteristics of the fast diatom currents most closely resemble those of vertebrate and invertebrate muscle Na+/Ca2+ currents. Conclusions/Significance This is the first demonstration of voltage-activated Na+ channels and the capacity to generate fast Na+-based action potentials in a unicellular photosynthetic organism. The biophysical and pharmacological characteristics together with the presence of a voltage activated Na+/Ca2+ channel homologue in the recently sequenced genome of the diatom Thalassiosira pseudonana, provides direct evidence supporting the hypothesis that this rapid signalling mechanism arose in ancestral unicellular eukaryotes and has been retained in at least two phylogenetically distant lineages of eukaryotes; opisthokonts and the stramenopiles. The functional role of the fast animal-like action potential in diatoms remains to be elucidated but is likely involved in rapid environmental sensing of these widespread and successful marine protists.