Effects of hydroxylamine and molybdate on the formation of nitrate reductase in the yeast Rhodotorula

NADPH-nitrate reductase (NR) and NADPH-cytochrome c reductase(CR) activities of Rhodotorula glulinis var. salinaria cellsgrown in nitrate medium supplied with hydroxylamine (0.2 mM)were respectively 1.6- and 3.1-fold higher than those of cellsgrown without hydroxylamine. NR formed in nitrate plus hydroxylaminemedium is almost totally in an inactive form which is reactivatedin vitro by K₃Fe(CN)₆. When molybdate (10^–7 M) was suppliedto this medium, total (active plus inactive) NR activity increasedfurther about twofold but CR activity somewhat decreased. Inordinary nitrate medium, such molybdate effects were small.Most of the CR derepressed (induced) excessively in the nitrateplus hydroxylamine medium had a molecular size similar to NRon the basis of Bio-Gel A 1.5 m chromatography. Some other propertiesof CR formed in this medium were the same as those of the CRmoiety of NR. Adding molybdate to the nitrate plus hydroxylamine medium aftergrowing the cells for 20 hr induced the development of NR activitywithout any increase in CR activity. This induction was completelyblocked by cycloheximide, puromycin and L-canavanine but notcompletely by 6-methylpurine. Ammonium repressed this inductionwith markedly decreasing CR activity. The roles of hydroxylamine and molybdate in the formation ofNR are discussed. (Received May 26, 1980; )     

Is Calmodulin Involved in Electrophysiology of Chara corallina?

The role of calmodulin in Chara was investigated using the antipsychoticdrug trifluoperazine (TFP), which is known to bind to the calmodulin/Ca⁺⁺complex preventing it from carrying out its normal functions. At low concentration (4.0 µM), TFP had profound and irreversibleeffects on the electrophysiology of Chara plasmalemma. The restingp.d. depolarized and the membrane conductance decreased suggestingan inhibition of the proton pump. After several hours of exposurethe membrane became leaky, as cells deteriorated. The excitation was also affected by TFP. Spontaneous repetitivefiring was observed. The excitation increased in duration andthe action potential peak depolarized to + 20 mV. The cytoplasmic streaming was unaffected by TFP; the streamingrate at the resting potential remained unchanged when TFP wasadded to the medium, stoppage occurred at the time of excitationand the streaming slowly resumed. Key words: Calmodulin, Chara corallina, Proton pump, Cytoplasmic streaming, Current, voltage characteristics     

Variation in the Structure and Response to Flooding of Root Aerenchyma in some Wetland Plants

The structure and response to flooding of root cortical aerenchyma(air space tissue) in a variety of wetland (flood-tolerant)species was investigated and compared with some flood-intolerantspecies. In some species aerenchyma consisted of enlarged schizogenousintercellular spaces and in others aerenchyma formation involvedlysigeny. Two types of lysigenous aerenchyma were distinguished.In the first the diaphragms between lacunae were arranged radiallyand consisted of both collapsed and intact cells. In the secondtype, which was confined to the Cyperaceae, the radial diaphragmscontained intact cells, and stretched between them were tangentially-arrangeddiaphragms of collapsed cells. Flooding in sand culture generally increased root porosity (airspace content) although there were exceptions. The flood-intolerantspecies Senecio jacobaea produced aerenchyma but did not survivelong-term flooding. Among the flood-tolerant species, Filipendulaulmaria did not produce extensive aerenchyma even when flooded.Eriophorum angustifolium and E. vaginatum produced extensiveaerenchyma under drained conditions which was not increasedby flooding. In Nardus stricta root porosity was increased bylow nutrient levels as well as by flooding. Aerenchyma, root cortex, wetland plants, waterlogging, flooding-tolerance, Ammophila arenaria, Brachypodium sylvalicum, Caltha palustris, Carex curia, Eriophorum vaginatum, Filipendula ulmaria, Glyceria maxima, Hieracium pilosella, Juncus effusus, Myosotis scorpioides, Nardus stricta, Narthecium ossifragum, Phalaris arundinacea, Senecio jacobaea, Trichophorum cespitosum     

Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca²⁺-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca²⁺ from internal stores,since influx of Ca²⁺ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa²⁺ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca²⁺ isconcluded to be caused by the release of Ca²⁺ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca²⁺ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994)     

Effect of Chilling Temperatures on the Protoplasmic Streaming of Plants from Different Climates

A microscope mount was designed so that specimen temperaturescould be monitored and controlled without impairing phase contrastoptics and used to measure rates of protoplasmic streaming between0 and 25 ?C in trichome cells of Lycopersicon esculentum, Lycopersiconhirsutum, Citrullus vulgaris, Tradescantia albiflora, Digitalispurpurea, and Veronica persica. Between 10 and 20 ?C the rates of streaming varied from 2–6µm s^–1 depending on the temperature, and differencesbetween the species were small. The temperature coefficientof streaming rates was found to increase as the temperaturewas lowered so that the plot of log rate against temperaturehad a steeper slope at the lower temperatures. The largest temperature cofficients were for the warmth-requiringL. esculentum (tomato) and C. vulgaris (water melon), and thesmallest for the temperate-zone plants V. persica (speedwell)and D. purpurea (foxglove). The changes in rate always occurredover a range of temperature; no ‘critical temperature’wasobserved below which streaming abruptly stopped and above whichit was active, although the amount of streaming as well as therate decreased as the lowest temperatures were approached. The temperatures experienced by the specimens during the experimentdid not affect the recovery of normal streaming rates betweenabout 10 and 20 ?C. In a population of a wild tomato, Lycopersicon hirsutum Humb.and Bonpl., collected from different altitudes in Peru and Ecuador,i.e. from locations of different environmental temperature,the rate of protoplasmic streaming at 5 ?C was greatest in thevarieties collected from the highest altitudes. The resultssuggest that streaming rates correlate with genetic adaptationto low temperature in the species examined.     

Basal Plate Tissue in Narcissus Bulbs and in Shoot Clump Cultures: Its Structure and Role in Organogenic Potential of Single Leaf Cultures

Light microscopy demonstrated that the apparently amorphous,achlorophyllous tissue at the base of in vitro shoot clump cultureof Narcissus was comparable in structure to the basal plateof Narcissus bulbs. Both had very complex vascularisation andsmall, densely packed parenchymatous cells. In shoot clump cultures, primordia were produced by meristematiczones at the surface of this achlorophyllous tissue, very closeto the base of leaves. Single leaf units excised from the invitro shoot clump cultures with a wedge of basal achlorophylloustissue were highly organogenic when used as secondary explantsfor in vitro culture of Narcissus. No organogenesis occurredin the absence of the leaf base and achlorophyllous (basal plate)tissue and little organogenesis occurred unless the leaf baseand basal plate tissue were immersed in the culture medium (i.e.explants inoculated into liquid medium or upright in agar-solidifiedmedium). After two 5-week culture passages in liquid medium, more thanfive leaves were produced per leaf base inoculated. Thus rapidmicropropagation of Narcissus can be achieved using only thebase of single leaf units excised from shoot clump cultures.Copyright1993, 1999 Academic Press Anatomy, basal plate, bulb, in vitro, leaf culture, Narcissus, organogenesis     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)     

Induction of Cytoplasmic Streaming and Movement of Chloroplast Induced by L-Histidine and its Derivatives in Leaves of Egeria densa

Rotational cytoplasmic streaming in leaves of Egeria densa wasinduced by light as well as by L-histidine (L-His). During bothtreatement with light and with L-His chloroplasts on the periclinalface were dislodged and moved to the anticlinal face where rotationalcytoplasmic streaming occurred. The effective concentrationof L-His was about 0.01 mM and the effect was almost saturatedat 0.1 mM. A derivative of L-His, 3-methyl-L-histidine, wasslightly less effective than L-His. By contrast, 1-methyl-L-histidinewas almost ineffective for induction of streaming, not onlyin Egeria but also in Vallisneria. Our resutlts are in markedcontrast to Fitting's result (1936) that 1-M-L-His is more effectivethan L-His. In Egeria, 1-methyl-L-His counteracted the stimulativeeffect of L-His. 1-Methyl-L-His penetrated into leaf cells ofEgeria to the same extent as 3-methyl-L-His and to a greaterextent than L-His. This observation excludes the possibilitythat the impermeability of leaves to 1-M-L-His might be responsiblefor its ineffectiveness. 1-M-L-His did not interfere with photodinesis.Differences in the mechanism of induction of rotational streamingby L-His and by light are discussed. ⁴ Present address: Fukui Institute of Technology, Gakuen, Fukui,910 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Intracellular Mobilization of Ca2+ and Inhibition of Cytoplasmic Streaming Induced by Transcellular Osmosis in Internodal Cells of Nitella flexilis

When transcellular osmosis was induced in internodal cells ofNitella flexilis that had been rendered inexcitable by treatmentwith KCl or EGTA, the rate of cytoplasmic streaming was reducedand the membrane was depolarized. In both KCl- and EGTA-treatedcells, the endoosmosis induced a significant increase in theconcentration of Ca²⁺ in the cytoplasm, which was demonstratedby monitoring the emission of light from aequorin that had beeninjected into the cytoplasm. When transcellular osmosis was induced in tonoplast-free cells,in which the intracellular Ca²⁺ concentration had been stabilizedat a very low level by treatment with the Ca²⁺-chelating agentEGTA, no change in the rate of cytoplasmic streaming on theendoosmosis side was observed. Hydration of the cytoplasm in the absence of endoosmosis wasinduced by direct introduction of a hypotonic medium into thevacuole by intracellular perfusion. The results mimicked theinhibition of streaming induced by transcellular osmosis. During transcellular osmosis, chloroplasts on the endoosmosisside swelled as a result of dilution of the cell sap. Swellingof chloroplasts was demonstrated to be unrelated to the inhibitionof streaming, since streaming was retarded at sites from whichchloroplasts had been removed. It is suggested that hydration of the cytoplasm triggers themobilization of Ca²⁺ from internal stores and causes an increasein the level of cytoplasmic Ca²⁺ that is responsible for theinhibition of streaming. Possible mechanisms for the osmosis-inducedincreases in the level of Ca²⁺ in the cytoplasm are discussed. (Received January 11, 1993; Accepted November 8, 1993)     

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Pearl Millet (Pennisetum americanum)

Immature embryos of Pennisetum americanum (pearl millet), culturedin the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) produceda pale-yellow and compact callus tissue by proliferation ofthe scutellum. Teased pieces of the compact callus were placedin a liquid medium on a gyrotory shaker to establish suspensioncultures. The cultures were composed of large, elongated andhigly vacuolated cells, and a population of richly cytoplasmiccells. The latter, here termed embryogenic cells, containednumerous plastids with starch, and occurred in tight groupsof four or more cells, and occasionally as single cells. Structuresresembling various stages of embryogenic development were foundin the suspension cultures. When the cultures were plated ina 2,4-D-free agar medium containing abscisic acid, embryoidswith the typical organization of cereal embryos were produced.The embryoids ‘germinated’ in vitro to give riseto plantlets, which were successfully transferred to soil. Theregenerated plants showed the normal diploid chromosome numberof 14. Embryoids apparently arose from single embryogenic cells,either directly or after the formation of a proembryonal massof cells. embryogenesis, pearl millet, Pennisetum americanum, regeneration, suspension culture     

Cytochalasin Rearranges Cortical Actin of the Alga Nitella into Short, Stable Rods

The cortical cytoplasm of the alga Nitella contains reticulateactin that does not survive perfusion fixation with glutaraldehydeunless prestabilized with the cross-linker 3-maleimidobenzoyl-N-hydroxysuccinimidester (MBS). Cytochalasin D remodels thiscortical actin into short rods which are more stable, survivingaldehyde fixation without MBS pre-treatment. The overall alignmentof these actin rods correlates with that of cortical microtubules(transverse in young cells, random in old cells) but probablydoes not involve one-to-one correspondence. The time course,dose dependence and reversibility of these structural changesbroadly resemble those for streaming inhibition by cytochalasinbut the cortical actin responds to concentrations that do notslow streaming. Because the structural changes concern the corticaland not the subcortical actin, they seem unlikely to directlyinhibit streaming. Formation of cortical rods is not a responseto streaming inhibition per se since it does not occur whentwo other inhibitors of streaming (2,4-dinitrophenol (DNP) andNethyl maleimide (NEM)) are used. NEM, however, resembles MBSin stabilizing the reticulate form of cortical actin even thoughit cannot cross link. ¹Address from July 1995; Department of Biology, Faculty of Science,Osaka University, Machikaneyama 1-1, Tayonaka, Osaka, 560 Japan.     

The Metabolic Fate of (4-Chloro-2-Methylphenoxy)Acetic Acid in Higher Plants: II. METABOLISM IN LIQUID CULTURED CALLUS CELLS OF WHEAT (Triticum aestivum L. )

Callus cells of two wheat cultivars in liquid medium rapidlyabsorbed (4-chloro-2-methylphenoxy)acetic acid (MCPA) and convertedit to ethanol-soluble products. The herbicide was transformedby methyl-hydroxylation, the major terminal residue being analcoholic glycoside of (4-chloro-2-hydroxymethylphenoxy)aceticacid. Minor metabolites were an ether-soluble conjugate of MCPA,an MCPA-glycoside and five additional aglycones. No metaboliteswere released into the medium. The rate of metabolism was lowerthan that of uptake such that a substantial amount of the radioactivityinitially accumulating in the cells was unmodified MCPA. Metabolismwas qualitatively and quantitatively similar to that in shootsexcised from seedlings. Cells also absorbed an ether-solubleconjugate of MCPA which had been isolated from carrot (Daucuscarota L. ), though less readily than MCPA itself. MCPA andthe above metabolites were produced, with the alcoholic glycosideagain as the major residue. Some MCPA was present in the medium,due probably to the action of extracellular hydrolases. Key words: MCPA, Triticum aestivum, Cell culture, Hydroxylation     

Variation in Tolerance and Metabolic Responses to Flooding in some Tropical Trees

The effect of flooding on aerobic and anaerobic respirationas well as on the internal levels of ethanol, lactic, succinicand malic acids were compared in three flood-tolerant and twonon-flood-tolerant species. In the non-flood-tolerant speciesKielmeyera coriacea and Pseudobombax marginatum, which comefrom the ‘ cerrado’ vegetation, there was a uniformityof response with ethanol being the only one of the above productsto accumulate substantially during flooding. In the flood-tolerantspecies, Sebastiana klotzchyana, Hymenaea courbaril var. stilbocarpaand Chorisia speciosa, flooding induced a variety of responseswhich indicate that the tolerant species have evolved differingstrategies to overcome flooding stress.     

Morphological Observations and Qualitative and Quantitative Studies of Auxins after Induction of Tobacco Genetic Tumor

When stem segments of tobacco F₁(Nicotiana glaucaxN. langsdorffii)seedlings were cultured on a hormone-free medium in the light,tumorous tissues were induced on the segments within 5 days.The fresh weight of the tissues increased rapidly, with a 10-foldincrease in weight every 6 days. Morphological observationsrevealed the active division of cells and the primordium-likestructures of the teratomas that had been induced during the5 days after cutting of the stem segments. In the light-growntissues of genetic tumors, IAA was revealed to be the predominantauxin by analysis by gas chromatography-mass spectrometry. NormalF₁ seedlings grown in the light contained endogenous IAA ata concentration of 3.4 ng/g fr wt. Five days after cutting,the level of endogenous IAA in stem segments rose to 96 ng/gfr wt or more, and then it decreased to 9.1 ng/g fr wt by 11days. This surge in the endogenous level of IAA may play animportant role in the induction of tobacco genetic tumor. (Received March 30, 1988; Accepted October 31, 1988)     

Isolation and Characterization of Temperature-Sensitive, High-CO2 Requiring Mutant of Anacystis nidulans R2

Temperature-sensitive (ts), high-CO₂ requiring mutants of Anacystisnidulans R2 were isolated by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) mutagenesis and ampicillin enrichment. One of these mutantswas able to grow under ordinary air enriched with 5% CO₂, butnot under ordinary air at 40?C. At 40?C, the concentration ofCO₂ at which the rate of oxygen evolution reached half the maximumvelocity (apparent Km(CO₂) in photosynthesis) was 1,000 timeshigher in mutant cells than in wild type cells, whereas therewas no significant difference in the maximum rate of photosynthesis. When wild type and mutant cells were incubated with 55 µmNaHC0₃ under illumination at 40?C, the initial rate of inorganiccarbon (IC) transport from the medium into the cells and themaximum internal IC accumulation were significantly higher inwild type cells than in mutant cells. These results indicate that the isolated high-CO₂ requiringmutant lacks the ability in transporting IC into the cells at40?C. Furthermore, the finding that the mutant cells which aredefective in IC transport cannot grow under ordinary air suggeststhe importance of IC concentrating system(s) in photosynthesisof cyanobacteria. Present address: Department of Molecular Biology, The Universityof Wyoming, Laramie, Wyoming 82071, U.S.A. To whom reprints should be requested. (Received April 28, 1988; Accepted September 14, 1988)     

Some Early Responses of Helianthus annuus L. to Flooding: I. THE EFFECTS OF FLOODING ON THE UPTAKE AND LEAKAGE OF 'NON-ELECTROLYTES' BY ROOTS

Drakeford, D. R., Mukherjee, I. and Reid, D. M. 1985. Some earlyresponses of Helianthus annuus L. to flooding. I. The effectsof flooding on the uptake and leakage of ‘non-electrolytes’by roots.—J. exp. Bot. 36: 1705–1715. The object of this work was to examine some of the early effectsof flooding on roots. A hydroponic system was developed thatgave good control over watering, degree of oxygenation of thebathing medium and allowed measurement of short term changesin the composition of the bathing medium. Excised roots, floodedfor 24 h, were shown to take up less [³H) ß-alaninethan non-flooded roots and also leaked more [³H] ß-alanineinto a distilled water bathing medium. Further, flooded excisedroots lost more protein to the bathing medium, with ‘young’(5–7 d) roots showing greater losses than ‘old’(11–14 d) roots. However, young roots had more proteinin the tissue even after greater loss. Young roots remainedhealthier and lost less fresh weight than old roots. Abscisicacid was shown to have a small role in protecting ‘young’roots from the effects of flooding. Key words: ABA, abscisic acid, anaerobic, flooding, leakage, roots, uptake, waterlogging     

Chlorolemma-A Continuous Membrane Layer between the Plasmalemma and the Streaming Cytoplasm in Nitellopsis obtusa and Nitella translucens, which takes part in the Generation of the Resting Potential, in its Light-Induced Changes and in the Generation of the Action Potential

At slow stepwise insertion of the glass micro-electrode intothe cell of Nitellopsis obtusa andNitella translucens four potentialjumps were recorded which corresponded to the penetration ofthe micro-electrode across the cell wall, the plasmalemma, thesupposed chlorolemma and the tonoplast, respectively. The photo-electricdepolarization appeared after the third jump only, which wassupposed to be the penetration of a membrane layer between theplasmalemma and the streaming part of the cytoplasm. The vibrationof this chlorolemma, caused probably by the protoplasmic streaming,resulted in large (up to 100 mV) hyperpolarizing and depolarizingpotential fluctuations when the micro-electrode tip was nearto this membrane. During cell excitation the potential difference(p.d.) had changed also across the chlorolemma- It was shownthat the potential changes across the chlorolemma, caused bylocal illumination on the isolated part of the cell, were measurablealso at a considerable distance (more than 1.4 cm) from theilluminated and isolated sequence. The light- and electron microphotographsobtained showed that the chloroplasts might really form a verycompact layer with tight connections between the neighbouringchloroplasts and that there existed at least one continuousmembrane layer which separated the immobile chloroplasts fromthe streaming cytoplasm. It was concluded that the p.d. measuredbetween the streaming cytoplasm and the external medium wasthe sum of the p.d.s across the cell wall, the plasmalemma andthe chlorolemma, respectively. Key words: Resting potential, Photo-electric responses, Characean algae, Chlorolemma     

Enzymatic Isolation of Protoplasts from Fern Protonemal Cells Stainable with a Fluorescent Brightener

Protoplasts were successfully isolated for the first time fromthe filamentous protonemal cells of ferns after the cells werecultured in contact with both air and medium. Sterilized sporesof Adiantum capillus-veneris and Pteris vittata were inoculatedon a piece of nylon mesh (40- {diaeresis}m mesh) placed on amat of polyester fibers which was soaked in liquid culture medium,and the spores were illuminated from above with continuous redlight. Protonemal cells, exposed to the air during this procedure,could be stained with Calcofluor White, a dye that binds tocell walls. Protoplasts were easily isolated from these protonemalcells by digestion of the cell wall with cellulase and pectinase.A total of 0.8–1.9 x 10⁴ and 0.6–2.0 x 10⁴ protoplastswere obtained from protonemata that originated from 10 mg ofdry spores of Adiantum and of Pteris respectively. Viability,as judged by staining with fluorescein diacetate was more than90% for both species. Staining with 4'6-diamidino-2-phenylindole(DAPI) revealed that about half of the protoplasts of both speciescontained a nucleus. (Received May 22, 1989; Accepted September 5, 1989)