Notes: The ecophysiology of the hypotonic response was studied in the charophyte alga, Lamprothamnium papulosum, which was grown in a marine (SW; 1072 mosmol kg–1) and a brackish (1/2 SW; 536 mosmol kg–1) environment. The cells produced an extracellular mucilage identified by histochemical staining as a mixture of sulphated and carboxylated polysaccharides. The thickness and chemical composition of the mucilage layer was a function of environmental salinity and cell age. Mucilage progressively increased in thickness from the apex (9 SW cells: 12·6 ± 1·8 μm; 15 1/2 SW cells: 4·8 ± 0·7 μm) to the base of the plants (15 SW cells: 44·8 ± 3·3 μm; nine 1/2 SW cells: 23·8 ± 2·5 μm); with a corresponding increase in the sulphated proportion. The mucilage was significantly thicker in SW plants. Hydraulic conductivity (Lp) at the apex of SW plants, measured by transcellular osmosis, was 8·3 × 10–13 m s–1 Pa–1. This was close to Lp of freshwater Chara (8·5 × 10–13 m s–1 Pa–1) which lacked mucilage. Basal SW cells with thicker mucilage had a smaller apparent Lp of 3·5 × 10–13 m s–1 Pa–1. The electrophysiology of the resting state and hypotonic response was compared in cells from the two environments based on current/voltage (I/V) analysis. The resting potential difference (PD) and conductance differed (11 SW cells: – 102·4 ± 10·1 mV, eight SW cells: 18·6 ± 2·4 S m–2; 19 1/2 SW cells: –125·7 ± 5·9 mV, 8·3 ± 0·8 S m–2). The type of cellular response to a hypotonic shock (decrease of 268 mosmol kg–1) also differed. In 1/2 SW plants, only the apical cells with thin mucilage responded classically with depolarization, conductance increase, Ca2+ influx, cessation of cytoplasmic streaming, and K+ and Cl– effluxes. Older cells making up the bulk of the plants responded with depolarization, but continued cytoplasmic streaming, and had only a small increase in conductance; or depolarized transiently without altering the I/V profile, conductance or streaming speed. Most cells remained depolarized and in the K+ state 1 h post-shock. Cells treated with the K+ channel blocker tetraethylammonium chloride also depolarized and remained depolarized. The SW cells depolarized but otherwise responded minimally to a 268 mosmol kg–1 drop in osmolarity and required a further 268 mosmol kg–1 down-step to elicit a change in the conductance. A spectrum of responses was measured in successively older and more mucilaginous cells from the same marine plant. We discuss the ecophysiological significance of the mucilage layer which modulates the cellular response to osmotic shock and which can be secreted to different degrees by plants inhabiting environments of different salinity.

Notes: Current-voltage (I/V) analysis and pharmacological dissection were applied to membranes of Lamprothamnium at the time of hypotonic stress. At least three types of process were found to be involved in the response to this stress.〈list xml:id="l1" style="custom"〉1The first 10min of exposure to hypotonic medium resulted in a depolarization of about 50mV accompanied by a decrease or no change in conductance. This depolarization occurred with either K+ or Ca2+ (and consequently C− channels inactivated.2The CI− channels opened mainly in the first 15min of the hypotonic stress, increasing the membrane conductance by about an order of magnitude.3The K+ conductance rose as the Cl− conductance started to diminish and reached a maximum after about 40 min.Both types of channel were strongly potential-dependent with a conductance peak between -150 and 0mV. An inactivation of K+or CI− channels resulted in moving the membrane potential away from the conductance maximum toward either EK or ECI, diminishing the ion efflux (and turgor regulation). The time courses of the conductance increases remained the same, suggesting that the conductance changes are not driven by feedback to some preset turgor level. The electrophysiology of the Lamprothamnium transporters is compared to that of salt-sensitive charophytes.

Notes: Cell-to-cell communication in lateral branches of limited growth in male Chara corallina plants has been studied using fluorescent plasmalemma-impermeable probes. The extent of cell-to-cell communication was found to be seasonal. In winter, branch internodes were characterized by relatively low plasmalemma potential differences (–121·2±23·2 mV; [K+]o 0·5 mol m−3; pH 7·6), quiescent dactyls isolated from the symplast (conic tips) and by restricted cell-cell communication (frequency of intercellular transport of six carboxyfluorescein = 42·9%). Cell-to-cell communication was inhibited during action-potentials, and was more extensive between cells which did not give an action potential in response to a current pulse. An inhibitor of the action potential, La3+, promoted cell-to-cell communication. These may be characteristic features of winter dormancy in lateral branches of Chara. In spring, morphological and electrophysiological changes occurred immediately prior to the onset of fertility. Characteristic changes in morphology included increased abundance of spherosomes and glycosomes, elongation of stipulodes and development of active branch dactyls. Spring branch inter-nodes were characterized by high plasmalemma PDs (–210·5±30·8mV; [K+]o 0·5 mol m−3; pH 7·6), predominantly active, non-isolated dactyls (spinaceous tips), and extensive intercellular communication (frequency of intercellular transport of 6 carboxyfluorescein = 89·2%). Artificial elevation of the intracellular calcium ion concentration by application of ionophore A23187 or Ca2+ microinjection significantly restricted intercellular communication to a level similar to that found in winter. The depression of intercellular communication in winter tissues is suggested to be due to high sensitivity to action potentials and/or to Ca2+ fluxes. Changes in intracellular Ca2+ distribution may be involved in the transition between the morphologically distinct states of dormancy and incipient fertility.

Notes: Cell-to-cell communication has been studied in lateral branches and developing antheridia of male Chara corallina plants. The moving cytoplasm is specialized to include essentially separate ascending and descending cytoplasmic streams within the inter-nodes. The neutral line which demarcates the ascending from the descending stream is established by the divisions of the nodal initial, which gives rise to both the node and internode. The ascending stream is located beneath the first-formed node-cells and the descending stream beneath the last-formed cells. The cells destined to develop into antheridia were always located on the same side as the descending internodal stream, and thus, were derived from the cells last formed during divisions of the nodal initial. Three stages of anther idial development have been defined: (1) young antheridia from the initial division of a node-cell to the formation of an octant structure; (2) maturing antheridia where differentiation into shield, manubria and capitular cells has occurred, including antheridia where an internal cavity has formed but contains filaments of less than 32 cells; and (3) mature antheridia where filaments contain more than 32 cells and spermatid production commences. Internodal cells of branches bearing young antheridia had similar characteristics to spring branches, including high plasmalemma potential differences (-217·7±31·5mV, [K+]o 0·5 mol m−3; pH 7·6) and extensive cell-to-cell communication (frequency of intercellular transport of 6 carboxyfluorescein 86%). The small probe 6 carboxy fluorescein moved into the entire young antheridium in 100% of injections. The molecular exclusion limit for internodes and the nodal complex lay between 874 and 1678Da whereas the exclusion limit for the young antheridium was smaller (between 750 and 874Da). Internodal cells of branches bearing maturing antheridia had similarly high PDs (–221·7±40mV; [K+]o 0·5 mol m−3; pH 7·6). Cell-to-cell communication between internodes bearing maturing antheridia was extensive (frequency of intercellular transport of 6 carboxyfluorescein 100%). The shield cells were isolated from the symplast of the thallus at this stage since they did not admit 6 carboxyfluorescein. Internodal cells of branches bearing only mature antheridia showed different characteristics. Intercellular communication between internodes was restricted to a level similar to that found in winter (frequency of intercellular transport of 6 carboxyfluorescein = 57%). The mature antheridium was entirely isolated from the symplast of the thallus. A period of extensive cell-to-cell communication and high PDs in internodal cells commences in vegetative lateral branches in spring, immediately before reproductive structures are initiated. These features persist throughout summer whilst reproductive structures develop, until the antheridial filaments contain 32 or more cells (mature stage), at which point spermatid production commences and the antheridium is isolated from the thallus. In autumn, following the stage of mature antheridia, no further antheridia are initiated. Internodes are subsequently vegetative throughout winter and their lateral branches are characterized by restricted cell-to-cell communication, low internodal PDs, and little obvious growth, all features consistent with winter dormancy.

Notes: Cells of the salt-tolerant charophyte Lamprothamnium respond differently to hypotonic challenge according to their position on the plant (i.e. cell age). Differences in electrophysiological response are coupled with differences in cell fine structure, and the presence or absence of extracellular mucilage. (1) Young, apical (fast-regulating, FR) cells respond with sudden cessation of cyclosis, depolarization to –50 mV (in some cells by more than 100 mV) and increase in membrane conductance by up to an order of magnitude. Intracellular [K+]v, [Na+]v and [Cl–]v decrease 1 h after hypotonic challenge. Patch-clamping cytoplasmic droplets reveals two types of K+ channel, 150 pS and 35 pS, and a small conductance Cl– channel, 35 pS (conductances at estimated tonoplast resting potential between zero and 20 mV). Extracellular mucilage is thin (〈 5 μm thick) or lacking, similar to freshwater Chara. Unlike freshwater charophytes these cells have a canalicular vacuolar system of large surface area and compartment the fluorochrome 6 carboxyfluorescein in the cytoplasm rather than the vacuolar system. (2) Older basal (slow-regulating, SR) cells do not cease streaming on hypotonic challenge and depolarize only slightly (by approximately 20 mV) with small or no change in membrane conductance. After 1 h the intracellular [K+]v, [Na+]v and [Cl–]v scarcely change. Patch-clamping cytoplasmic droplets reveals two types of K+ channel, medium conductance 90 pS and low conductance (as in FR cells). The large conductance K+ channel was not observed. The Cl– channel was more active in SR cells. The cells were coated with extracellular mucilage more than 10 μm thick. In a similar manner to freshwater Chara, these cells compartment 6 carboxyfluorescein in a large central vacuole. In the older cells, making up the bulk of any given plant, the simultaneous development of extracellular mucilage and a large central vacuole which compartments 6 carboxyfluorescein is associated with a minimal electrophysiological response to hypotonic challenge. The significance of these findings for salt-tolerance is discussed.