Apical growth of protonemata inAdiantum capillus-veneris

Action spectra for the induction of apical swelling in red-light-grown single-celled protonemata of the fernAdiantum were determined by continuous irradiation with monochromatic light for 5 hr. The resultant action spectra showed a sharp peak at 480 nm with a broad plateau in the region of blue and near ultraviolet light. Wave-lengths longer than 520 nm had no effect. When the tips of filamentous protonemata were irriadiated with a narrow beam (20 μm in width) of blue light for 3 hr, apical swelling and apical growth inhibition obviously took place in all protonemata tested, while no significant effect was observed when any other regions than the tip were irradiated. Polarized blue light vibrating parallel with the developmental axis of protonemata induced apical swelling and also prevented apical growth as effectively as non-polarized light, but that vibrating in a normal direction was significantly less effective.     

Effects of simulated microgravity on the germination and elongation of protonemata of Adiantum capillus-veneris

Germination of spores and elongation of protonemata of Adiantum capillus-veneris L., which can be controlled by light irradiation, were examined under various gravitational conditions including microgravity simulated by a three-dimensional clinostat. The elongation of protonemata that had been irradiated from below and grew downwards was greater than that of protonemata growing horizontally or upwards. Under microgravity, protonemata were shorter than the controls. Germination of spores, direction of growth, and cell division were not affected by gravitational conditions.     

Effects of hypergravity on the elongation and morphology of protonemata of Adiantum capillus-veneris

Elongation growth of protonemata of Adiantum capillus-veneris , which can be controlled by light irradiation, was examined under acropetal and basipetal hypergravity conditions (from -13 to +20 g ) using a newly developed centrifugation equipment. Elongation of the protonemata under red light was inhibited by basipetal hypergravity at more than +15 g but was promoted by acropetal hypergravity from -5 to -8 g . Division of the protonemal cells that was induced by white light was inhibited under basipetal hypergravity at +20 g but was unaffected under acropetal hypergravity at -15 g . Upon exposure to continuous red light for 7 to 8 days, most of the protonemata grew as filamentous cells in the absence of a change in the normal gravitational force (control), but more than half of the protonemal cells were abnormal in terms of shape when maintained under hypergravity at +20 g .     

The mechanism of auxin uptake and accumulation in moss protonemata

Protonemata of the moss Funaria hygrometrica Sibth. (L.) show a strong pH dependence for auxin accumulation. IAA uptake is enhanced when the pH of the incubation medium is lowered from 7.6 to 4. In low light intensity grown protonemata (0.56 W m⁻²) a component of IAA uptake could be saturated by IAA; efflux of IAA could be inhibited by 2,3,5-triiodobenzoic acid. This is explained by the presence of influx and efflux carriers for IAA. In protonemata grown at high light intensity (2.00-2.30 W m⁻²) these carriers could not be shown to be present. These results are discussed with regard to the different physiological behavior of moss protonemata grown under different light conditions.     

The Protonema of Chara fragilis Desv.: Regenerative Formation,Photomorphogenesis, and Gravitropism

When exposed to constant white light for four weeks, isolated nodes of Chara fragilis Desv. regenerated side branches, rhizoids, and multicellular protonemata, the latter being similar to those germinated from oospores. When kept in darkness the nodes developed protonemata exclusively. These were single-celled, colourless, and tip-growing and, with the light microscope, they looked like rhizoids. Upon exposure to blue light, but not to red or far-red, the growth rates of the protonemata rapidly declined, the cell apices swelled, and the nucleus migrated acropetally. Within 24 h the cells went through the first of a series of divisions resulting in the formation of multicellular protonemata. When returned to darkness after a blue light pulse of 5 h the cell divisions proceeded normally, but the protonemata showed etiolated growth. While growth of the internode was drastically promoted, the development of the multicellular apex and the lateral initial were suppressed. Both uni- and multicellular etiolating protonemata showed negative gravitropism but were phototropically insensitive. It is argued that the single-celled protonema is an organ specialized for the penetration of mud covering the nodes or oospores of Chara and thus serves to search for light, comparable to etiolated hypocotyls and stems in seedlings of higher plants.     

BIOASSAY OF ANTHERIDIOGEN IN LYGODIUM JAPONICUM

Antheridia were induced by exogenously applied GA₃ at concentrations between 10⁻⁶ and 3 × 10⁻⁴ M in very young filamentous protonemata of Lygodium japonicum grown in darkness; the longer the dark preculture of protonemata, the lower was the sensitivity of the protonemata to GA₃. Antheridial initials were discernible after 36 hr of GA₃ treatment in the most sensitive protonemata, and the timing of antheridial initiation was delayed with increasing protonemal age.
This quantitative response of the protonemata provided the basis for a new method of assaying gibberellins in terms of the degree of antheridial formation. According to this method, all the gibberellins tested and one of their precursors were active in inducing antheridia in the protonemata, and the activity spectrum of the gibberellins was as follows: GA₇>GA₄>GA₉>GA₃>GA₅>GA₁>GA₈.
The amounts of antheridiogen contained in conditioned media were measured by the present bioassay. A semi-logarithmic relation was shown between the percentage of antheridial formation and the concentration of conditioned medium within a certain dilution range. The amounts of antheridiogen secreted by the prothallia were quantitatively compared by transferring samples onto fresh media for a short period of time.     

Quantitative imaging of directional transport through plasmodesmata in moss protonemata via single-cell photoconversion of Dendra2

Cell-to-cell transport of molecules in plants must be properly regulated for plant growth and development. One specialized mechanism that plants have evolved involves transport through plasmodesmata (PD), but when and how transport of molecules via PD is regulated among individual cells remains largely unknown, particularly at the single-cell level. Here, we developed a tool for quantitatively analyzing cell-to-cell transport via PD at a single-cell level using protonemata of Physcomitrella patens and a photoconvertible fluorescent protein, Dendra2. In the filamentous protonemal tissues, one-dimensional intercellular communication can be observed easily. Using this system, we found that Dendra2 was directionally transported toward the apex of the growing protonemata. However, this directional transport could be eliminated by incubation in the dark or treatment with a metabolic inhibitor. Thus, we propose that directional transport of macromolecules can occur via PD in moss protonemata, and may be affected by the photosynthetic and metabolic activity of cells.     

中华缩叶藓孢子萌发与原丝体发育特征研究

通过室内人工培养中华缩叶藓的孢子,在光学显微镜下详细观察了其孢子萌发、原丝体发育及配子体发生的全过程.结果表明:中华缩叶藓的孢子在壁内萌发,随后分裂产生块状原丝体;块状原丝体上可产生两种丝状体,一种是具疣的棒状原丝体,另一种是由长圆柱状细胞组成的轴丝体;配子体原始细胞只产生于块状原丝体上.根据中华缩叶藓的孢子萌发和原丝体发育特征,并参照Nishida对藓类植物孢子萌发类型的划分,确定中华缩叶藓的萌发孢子型应属于缩叶藓型(Ptychomitrium-type).     

Actomyosin-mediated statolith positioning in gravisensing plant cells studied in microgravity

The positioning and gravity-induced sedimentation of statoliths is crucial for gravisensing in most higher and lower plants. In positively gravitropic rhizoids and, for the first time, in negatively gravitropic protonemata of characean green algae, statolith positioning by actomyosin forces was investigated in microgravity (<10(-4) g) during parabolic flights of rockets (TEXUS/MAXUS) and during the Space-Shuttle flight STS 65. In both cell types, the natural position of statoliths is the result of actomyosin forces which compensate the statoliths' weight in this position. When this balance of forces was disturbed in microgravity or on the fast-rotating clinostat (FRC), a basipetal displacement of the statoliths was observed in rhizoids. After several hours in microgravity, the statoliths were loosely arranged over an area whose apical border was in the same range as in 1 g, whereas the basal border had increased its distance from the tip. In protonemata, the actomyosin forces act net-acropetally. Thus, statoliths were transported towards the tip when protonemata were exposed to microgravity or rotated on the FRC. In preinverted protonemata, statoliths were transported away from the tip to a dynamically stable resting position. Experiments in microgravity and on the FRC gave similar results and allowed us to distinguish between active and passive forces acting on statoliths. The results indicate that actomyosin forces act differently on statoliths in the different regions of both cell types in order to keep the statoliths in a position where they function as susceptors and initiate gravitropic reorientation, even in cells that had never experienced gravity during their growth and development.     

Effects of hardening and freezing stress on membrane lipids and CO2 fixation of Ceratodon purpureus protonemata

Membrane lipids and steady-state CO₂ fixation rates were studied in moss protonemata in order to evaluate separately the effects of growth temperature, freezing stress and the achievement of frost hardiness. Protonemata of Ceratodon purpureus (Hedw.) Brid, were grown at 20 and 4°C and parts of both materials were then hardened. The low growth temperature increased the content and unsaturation level of membrane lipids significantly. This did not, however, cause a noticeable increase in the frost hardiness. Nor was the achievement of frost hardiness in this material accompanied by further changes in the amount or unsaturtion level of any membrane lipid class. Cytoplasmic membranes were abundant in both unhardened and hardened materials grown at 4°C, which agreed with the high phospholipid content of these protonemata. The only significant difference in membrane lipids between unhardened and hardened materials was a 50% lower level of trans 16:1 fatty acid in the phosphatidylglycerol fraction of hardened protonemata.
In hardened protonemata monogalactosyldiacylglycerol (MGDG) was the membrane lipid most liable to decrease during the freeze-thaw cycle. The loss of MGDG was accompanied by partial inhibition of CO₂ fixation. Provided the content of phospholipids remained unchanged (freeze-thaw cycle with – 10°C in hardened protonemata), this inhibition was mostly reversible. If loss of the phospholipids also had occurred during the freeze-thaw cycle, as was the case in unhardened material at or below -10°C, CO₂ fixation was severely and nearly irreversibly inhibited after thawing.     

Hypergravity can reduce but not enhance the gravitropic response ofChara globularis protonemata

Summary The relationship between the position of the statoliths and the direction and rate of tip growth in negatively gravitropic protonemata ofChara globularis was studied with a centrifuge video microscope. Cells placed perpendicularly to the acceleration vector (stimulation angle 90 °) showed a gradual reduction of the gravitropic curvature with increasing accelerations from 1g to 8g despite complete sedimentation of all statoliths on the centrifugal cell flank. It is argued that the increased weight of the statoliths in hypergravity impairs their acropetal transport which is induced when the cell axis deviates from the normal upright orientation. When the statoliths were centrifuged deep into the apical dome at 6g and a stimulation angle of 170 ° the gravitropic curvature after 1 h was identical to that determined for the same cells at 1g and the same stimulation angle. This indicates that gravitropism in Chara protonemata is either independent of the pressure exerted by the statoliths on an underlying structure or is already saturated at 1g. When the statoliths were moved along the apical cell wall at 8g and the stimulation angle was gradually increased from 170 ° to 220 ° the gravitropic curvature reverted sharply when the cluster of statoliths passed over the cell pole. This experiment supports the hypothesis that in Chara protonemata asymmetrically distributed statoliths inside the apical dome displace the Spitzenkörper and thus the centre of growth, resulting in gravitropic bending. In contrast to the positively gravitropic Chara rhizoids, no modifications either in the transport of statoliths during basipetal acceleration (6g, stimulation angle 0 °, 5 h) or in the subsequent gravitropic response could be detected in the protonemata. The different effects of centrifugation on the positioning of statoliths in Chara protonemata and rhizoids indicate subtle differences in the function of the cytoskeleton in both types of cells.Dedicated to Prof. Dr. Zygmunt Hejnowicz on the occasion of his 70th birthday     

Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

Membrane lipids in Ceratodon purpureus protonemata grown at high and low temperatures

Ceratodon purpureus (Hedw.) Brid. was grown at two temperatures, 20 and 4°C. The protonemata grown at 4°C fixed more CO₂ at low temperatures; but their frost tolerance, tested as the recovery of photosynthesis after frost treatment, was not better than in the protonemata grown at 20°C. The effects of the growth temperature were studied on the membrane lipids of intact protonemata and on the lipid and protein contents of isolated thylakoid membranes. A large proportion, 70 to 90%, of the thylakoid membrane lipids was lost unless precautions were taken to inhibit the lipid-degrading enzyme activities. The lipid content of the thylakoid membranes of protonemata grown at 20 and 4°C was 3.9 and 4.8 mol (mol chlorophyll)⁻¹, respectively. Only minor differences were found in the lipid class composition. Monogalactosyldi-acylglycerol constituted more than 50 mol-% of the thylakoid membrane lipids at both 20 and 4°C. However, each lipid class had a higher average number of double bonds per lipid molecule in cold growth conditions. The protein content of the thylakoid membranes was low at both 20 and 4°C. These characteristics of the thylakoid membranes may be a prerequisite for the observed ability of protonemata to photosynthesize even at subzero temperatures.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

Gravitropism in caulonemata of the moss Pottia intermedia

The gravitropism of caulonemata of Pottia intermedia is described and compared with that of other mosses. Spore germination produces primary protonemata including caulonemata which give rise to buds that form the leafy moss plant, the gametophore. Primary caulonemata are negatively gravitropic but their growth and the number of filaments are limited in the dark. Axenic culture of gametophores results in the production of secondary caulonemata that usually arise near the leaf base. Secondary protonemata that form in the light are agravitropic. Secondary caulonemata that form when gametophores are placed in the dark for several days show strong negative gravitropism and grow well in the dark. When upright caulonemata are reorientated to the horizontal or are inverted, upward bending can be detected after 1 h and caulonemata reach the vertical within 1-2 d. Clear amyloplast sedimentation occurs 10-15 minutes after horizontal placement and before the start of upward curvature. This sedimentation takes place in a sub-apical zone. Amyloplast sedimentation also takes place along the length of upright and inverted Pottia protonemata. These results support the hypothesis that amyloplast sedimentation functions in gravitropic sensing since sedimentation occurs before gravitropism in Pottia and since the location and presence of a unique sedimentation zone is conserved in all four mosses known to gravitropic protonomata.     

Gravity perception requires statoliths settled on specific plasma membrane areas in characean rhizoids and protonemata

Summary. The noninvasive infrared laser micromanipulation technique (optical tweezers, optical trapping) and centrifugation were used to study susception and perception, the early events in the gravitropic pathway of tip-growing characean rhizoids and protonemata. Reorientation of the growth direction in both cell types was only initiated when at least 2–3 statoliths settled on specific areas of the plasma membrane. This statolith-sensitive plasma membrane area is confined to the statolith region (10–35 μm behind the tip) in positively gravitropic rhizoids, whereas in negatively gravitropic protonemata, this area is limited to the apical plasma membrane (0–10 μm). Statolith sedimentation towards the sensitive plasma membrane areas is mediated by the concerted action of actin and gravity. The process of sedimentation, the pure physical movement, of statoliths is not sufficient to initiate graviresponses in both cell types. It is concluded that specific statolith-sensitive plasma membrane areas play a crucial role in the signal transduction pathway of gravitropism. These areas may represent the primary sites for gravity perception and may transform the information derived from the gravity-induced statolith sedimentation into physiological signals which trigger the molecular mechanisms of the opposite graviresponses in characean rhizoids and protonemata. Received September 10, 2001 Accepted November 16, 2001     

Succession of soil algae on dumps from strip coal-mining in the Most region (Czechoslovakia)

In the years 1985–1986, the primary succession of algae on dumps from brown coal mining was studied in the Most region (North Bohemia, Czechoslovakia). The colonization of sterile clayic substrates by algae (several coccal species of chlorophytes and heteroconts) and mosses (protonemata) starts before the first ecesis of higher plants (in deposits about 3 months old). Diatoms and, later, cyanophytes accompany the algal community after 1 year. Filamentous types of heteroconts and green algae occur first on 7 year old dumps. Green algae represent the commonest group throughout the succession. The number of species continuously increases with the age of the dumps and finally reaches about 40 species from the 18th to 30th year. The species composition is similar to that in grassland biotopes in Czechoslovakia.     

Tip-growing cells of the moss Ceratodon purpureus Are gravitropic in high-density media

Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm(-3). Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.