Actin filament array during side branch initiation in protonema cells of the mossFunaria hygrometrica: an actin organizing center at the plasma membrane

Summary With an improved method to visualize the actin filament system it is possible to detect a small, peculiar accumulation of actin filaments under the prospective area of side branch formation inFunaria protonema cells. It consists of a ring-like configuration of actin filaments from which filaments radiate, preferentially along the plasma membrane. During the transition to tip growth the arrangement becomes loosened and eventually disappears whereas the filaments are concentrated in inner regions of the cytoplasm with a maximum in the apical dome.     

Scapania praetervisa Meyl., not S. mucronata Buch in Britain

Abstract

The protonema of Orthotrichum obtusifolium, regenerated from leafy shoots, comprises a dense mass of largely unbranched exclusively upright filaments. All the cells have transverse cross-walls and contain numerous chloroplasts which become larger and discoidal in older filaments. Differentiation into chloro- and caulonema is absent. When upright filaments are transplanted onto an agar surface, they act as the main axes for a further set of upright branches. Buds and side branches are produced symmetrically at right angles to the mother cells. Protonemal morphogenesis hardly changes on nutrientfree medium and spherical brood cells are not produced after prolonged culture or in the presence of abscisic acid. Rhizoids with oblique septa and pigmented walls arise from the base of gametophores. These revert to protonemata when in contact with nutrient agar.

The upright filaments produce terminal and intercalary filamentous gemmae with well defined abscission cells. The latter swell and lose their contents prior to liberation. Cells immediately below the abscission cells produce side branches in acropetal succession. Secondary gemmae arise by percurrent proliferation and gemmae frequently germinate in situ.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Segregative cell division and the cytoskeleton in two species of the genus Struvea (Cladophorales,Ulvophyceae, Chlorophyta)

The detailed segregative cell division (SCD) processes and changes in the arrangement of cortical microtubules and actin filaments were examined in two species of Struvea. SCD was initiated by the appearance of annular constrictions along the lateral side of a mother cell. The constrictions decreased in diameter, became thin, tubular in shape, and pinched the protoplasm of the mother cell into several protoplasmic sections. The protoplasmic sections expanded and developed into daughter cells, which appressed each other, and were arranged in a single row. Lateral branches protruded from the upper parts of the daughter cells. The protoplasm of the lateral branches was divided by secondary SCDs and was distributed amongst the new daughter cells. SCD and lateral branch formation were essential for morphogenesis in Struvea. Cortical microtubules were arranged parallel and longitudinally to the cell axis before SCD. When SCD was initiated, there was considerable undulation of the cortical microtubules and several transverse bundles appeared in the cytoplasmic zone where annular constrictions occurred. A microtubule‐disrupting drug (amiprophos methyl) inhibited SCD. Actin filaments maintained reticulate patterns before and during SCD. These results demonstrated that SCD in Struvea species was quite distinct from that in Dictyosphaeria cavernosa reported previously.     

Germination of the Spores and Development of Primary and Secondary Protonema of Funaria hygrometrica

Abstract

1. The primary protonema of Funaria hygrometrica, cultivated on Knop's or Marchal's agar in the light, proved to consist of filaments with much chlorophyll, a hyaline membrane, perpendicular cross-walls and branches equal to the main filament (chloronema). These filaments grow on the surface of the agar, the branches may also grow vertically. Sometimes filaments with less chlorophyll occur immediately after the germination. The caulonema described by Sironval has not been observed. Thus the rhizoid-like forms mentioned in the literature should more likely be considered as a result of external conditions (see Schoene, Bauer, Heitz and Fitting). Therefore it remains doubtful if a distinction between rhizoids and chloronema on the primary protonema is of any importance as it is impossible to give a good definition of either form.

At the base of moss plants main filaments with brown membranes, oblique septa and without chlorophyll may develop (rhizoids). They grow on the surface or within the agar. In F. hygrometrica especially, the stem seems to influence the occurrence of these rhizoids. The main filaments form buds on the basal cell of the branches and thus serve for vegetative reproduction. The branches show the characteristics of the chloronema. This is contrary to the conclusion of Westerdijk that rhizoids would pass into chloronema only when they are damaged or when the growth of the end bud of the plant is inhibited. At the base of the plant, moreover, little ramified, short branches with oblique septa appear which do not produce buds.

2. Branches may develop in the first growth stages of the primary protonema at any point of the cells. One single cell of a main filament can produce none, one, or more than one branch. Later the branches appear immediately behind the acroscopic cross-wall except in a few cases. Each cell then produces one branch.

3. Buds always develop at the basal cell of a primary branch of a green main filament or of a rhizoid derived from a moss plant.

4. In two ways the protonema may fall into pieces, which can develop into new main filaments:

(a) By forming brood cells; rounded cells which get detached by splitting of the septum. This phenomenon is very frequent. Contrary to Servettaz's opinion it seems to occur particularly under unfavourable conditions.

(b) By forming special cells, tmemata, whose walls are rent. These occur on the primary protonema contrary to the observations of Correns and Bauer, but they are much less frequent than the brood cells. No observations have been made on the circumstances of their occurrence.     

The reproductive morphology of Dumontia incrassata (O. F. Müller) Lamouroux

The reproductive morphology of Dumontia incrassata (O. F. Müller) Lamouroux is described from New Hampshire, U.S.A. populations. Mature carpogonial and auxiliary branches are usually 5 and 6 celled, respectively, but both may range from 4 to 9 cells. Auxiliary branches are more numerous than carpogonial branches; however, the ratio of carpogonial to auxiliary branches increases towards the apex of the frond. A linear relationship exists between the position of the generative auxiliary cell and the number of cells in the auxiliary branches. In most cases the third cell from the top of the auxiliary branch becomes the generative auxiliary cell. The probabilities for different positions of the generative auxiliary cells are summarized. During spermatangial development, four mother cells are initially produced per cortical cell initial; subsequently, successive spermatangia are cut off obliquely and on opposite sides of each mother cell. Monoecious asexual plants of D. incrassata are described, as well as an irregular tetraspore development.Scientific Contribution Number from the New Hampshire Agriculture Experiment Station; also issued as contribution No. 81 of the Jackson Estuarine LaboratoryScientific Contribution Number from the New Hampshire Agriculture Experiment Station; also issued as contribution No. 81 of the Jackson Estuarine Laboratory     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Experiments with inhibitors and with centrifugation

Summary Colchicine treatment ofFunaria caulonemata, usually does not inhibit initiation of a side branch or its incipient elongation but does prevent movement of chloroplasts and the nucleus into the outgrowth. After colchicine and after cytochalasin B treatment side branches are formed about at the normal age of the cells; because of the inhibition of the apical cell they arise at an abnormal position,i.e., not in the third but in the second cell of a filament. After D₂O treatment the organelles are dislocated toward the basal cross wall. The site of side branch formation is then obviously determined by the position of the nucleus. Cells with an irreversibly reversed longitudinal polar axis can be found; by centrifugation in proximal direction the sites of side branch initiation likewise are displaced into the proximal region of the cell, especially if the remigration of the nucleus is inhibited by colchicine. High concentrations of Ca²⁺ ions induce the formation of side branch cells, without any outgrowth. The calcium ionophore A 23 187 influences the position of the nucleus and of the side branch only slightly. After these various treatments intercalary divisions frequently occur. The role and interrelationship of the nucleus and peripheral cytoplasm in establishing and maintaining the polar axes, and the role of microtubules are discussed.     

Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex

The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-P(i) filaments than on ADP-actin filaments.     

Studies on the Development of the Reproductive Organs of Caloglossa leprieurii (Mont.) J. Ag.

The cell of Caloglossa leprieurli is a polycaryon. The sexual thalli are usually dioecious. Its life cycle involves the alternation of three generations. In the reproductive season, the appearance of tetrasporophyte and female and male gametophyte shows evident difference. The tetrasporophyte is big and flat. The branches are sparse. Many small red spots, i.e. tetrasporogial groups, can be seen with the naked eyes in the upper part of the branch. The terminal part of the female gametophyte is a little twisted with thick branches. The cystocarps are spherical and most of them are located in ventral side of the upper branch points. The male gametophyte is smaller with less branches. Its terminal part is a little twisted with pale colour. The pit connections of the vegetative cells of every thallus are all well developed. The carpogonial branch consists of four cells. Generally, it is formed by the division of the pericentral cell. After fertilization of the carpogonium, an auxiliary cell is formed by the division of supporting cell. Usually five to seven, even more than ten young procarps can be formed at the apical part of the reproductive branch. The procarps often occur on consecutive segments of pericentral ceils. But among most of them only a carpogonium is fertilized and developes into cystocarp. When a carpogonium is fertilized, the other one on this branch usually cease further development. The spermatangia are formed on the lateral wing cells of both sides of the upper part midrib of a reproductive branch and distributed on both dorsal and ventral surfaces. A vegetative cell is divided into three cells in parallel to the surface of the thallus. The cortical cells under the two outer surfaces forms three to four permatangial mother cells. Each spermatangial mother cell divides into two to four spermatangia. After the sperm dispersal of the mature spermatangia, the spermatangial mother cells are still retained on both sides of the middle discoid cells.     

鹧鸪菜生殖器官发育的研究

鹧鸪菜[Caloglossa leprieurii(Mont)J.Ag.]细胞含多核。在生殖季节,孢子体、雌、雄配子体在外形上有明显的差异:四分孢子体大而平展,分枝的上部肉眼可见有红色小斑点,即为四分孢子囊群。雌配子体顶部略蜷曲,分枝密,囊果呈球状,多位于分枝上部分枝点的腹面。雄配子体个体较小,枝顶蜷曲,颜色淡,特别生有精子囊的部分更淡。每种藻体的营养细胞的纹孔连结非常发达,每个细胞都具有4-5条,多者达7-8条。果胞枝由四个细胞组成,一般由围轴细胞分裂形成,果胞受精后,由支持细胞分裂形成辅助细胞,一个生育小枝上,可以连续形成5-7个甚至10个以上幼果胞系,但多数仅有一个果胞受精发育成囊果。当一个果胞受精时,其他果胞系就停止发育。精于囊位于分枝上部中肋的两侧,背腹面均有分布。一个营养细胞沿着叶面平行方向平周分裂成三个细胞,在两个外侧细胞各形成3-4个精子囊母细胞,每个精子囊母细胞斜壁分裂产生2-4个精子囊,放散后,余下的精子囊母细胞仍留在中间盘状细胞上。     

Four distinct photoreceptors contribute to light-induced side branch formation in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Side branch formation in the moss, Physcomitrella patens, has been shown to be light dependent with cryptochrome 1a and 1b (Ppcry1a and Ppcry1b), being the blue light receptors for this response (Imaizumi et al. in Plant Cell 14:373, 2002). In this study, detailed photobiological analyses were performed, which revealed that this response involves multiple photoreceptors including cryptochromes. For light induction of branches, blue light of a fluence rate higher than 6 μmol m⁻² s⁻¹ for period longer than 3 h is required. The number of branches increased with the increase in fluence rate and in the irradiation period. The number of branches also increased when red light was applied together with the blue light, although red light alone had a very few effect. By partially irradiating a cell, both receptive sites for blue and red light were found to be located around the nucleus. Further, both red and blue light determine the positions of branches being dependent upon the vibration plane of polarized light. Red light control of branch position was nullified by simultaneous far-red light irradiation. A blue light effect on branch position was not found in lines with disrupted phototropin genes. Thus, dichroic phytochrome and phototropin, possibly on the plasma membrane, regulate branch position. These results indicate that at least four distinct photoreceptor systems, namely, cryptochromes and red light receptor around or in the nucleus, dichroic phytochrome and phototropin around the cell periphery, are involved in the light induction of side branches in the moss Physcomitrella patens.     

A MORPHOLOGICAL STUDY OF HOMMERSANDIA MAXIMICARPA GEN. ET SP. NOV. (KALLYMENIACEAE,RHODOPHYTA) FROM THE NORTH PACIFIC1

A new foliose red alga, common subtidally from British Columbia to the Aleutian Islands, is described and given the name Hommersandia maximicarpa. The lobed perennial thallus, which can reach a height of 23 cm, is distinguished by its vegetative structure and by its unique pattern of nonprocarpic carposporophyte development. In transverse section, the blades consist of a narrow filamentous medullary layer sandwiched on either side by large ellipsoidal subcortical cells and a thin outer cortex. The monocarpogonial branch and auxiliary cell systems of the female plants are typical of many members of the Kallymeniaceae. However, after the carpogonialfusion cell forms, a distinctive developmental pattern begins. The connecting filaments radiate outward into the surrounding tissue, branch abundantly, and become septate. They then contact, in addition to auxiliary cells, many small moniliform accessory branches. These branches appear to act as initiation points for the gonimoblast filaments. The large diffuse carposporophytes produced are unknown in any other member of the Cryptonemiales. The vegetative and reproductive anatomy of Hommersandia is compared to other Kallymeniaceae, and similar patterns of postfertilization development are examined in the Rhodophyta.     

Effects of nifedipine,verapamil, and diltiazem on tip growth inFunaria hygrometrica

Protonemata ofFunaria hygrometrica Sibth. were treated with nifedipine, verapamil, or diltiazem. Responses to each of the drugs were, on the one hand, reduction of growth rate and tip cell length and, on the other hand, formation of apical swellings in caulonema tip cells and of anomalously oriented separation walls between main filaments and young side branches. The first effect is regarded as a more general expression of inhibition while the second complex of effects is attributed to perturbations in directed vesicle transport. Replacement of drug-containing media by normal Knop agar demonstrated the reversibility of inhibitor action: growth parameters were comparable to those of control protonemata within a few hours. A fast reaction, the formation of subapical vacoules, occurred within minutes of drug application and was only observed with verapamil and diltiazem. In connection with this process, rapid migrations of chloroplasts took place, but examination of the microtubule cytoskeleton in such cells by indirect immunofluorescence with a monoclonal antibody against tubulin showed an intact microtubule network. callose deposits in tip cells treated with verapamil. They were polarly distributed and started to appear in cell apices about 2h after the beginning of verapamil application. Two mechanisms of action for the tested inhibitors are discussed: (i) perturbations of membrane permeability by interference with one or more of the cell's Ca²⁺-transport systems, and (ii) a more indirect mechanism affecting vesicle transport via the microfilament system.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Connection between the Synthesis of Differentiation Specific Proteins and the Capacity of Cells to Respond to Cytokinin in the Moss Funaria

The differentiation stage of the caulonema in Funaria hygrometrica protonema is distinguished from the chloronema stage by three additional protein bands (CSP) in the soluble protein fraction. During the change of caulonema to chloronema, which is induced by isolation of single filaments (regeneration), the CSP disappear. This is not the consequence of an accelerated degradation or turnover but of a gradual termination in the de novo synthesis of CSP during regeneration as demonstrated by pulse-chase experiments with l -[4,5–³H] leucine. Cytokinin inhibits the termination of the synthesis of CSP. The decrease in synthesis parallels the decrease in ability of the isolated caulonema cells to respond to cytokinin via bud formation. It is therefore concluded that the CSP are involved in the competence of caulonema cells to respond to cytokinins.     

Innervation pattern of the gill arches and gills of the carp (Cyprinus carpio)

The innervation pattern of the respiratory gill arches of the carp (Cyprinus carpio) is described. The gill region is innervated by the branchial branches of the glossopharyngeal and vagal nerves. Each branchial nerve divides at the level of or just distal to the epibranchial ganglion into: 1) a pretrematic branch, 2) a dorsal pharyngeal branch, and 3) a posttrematic branch. The dorsal pharyngeal branch innervates the palatal organ in the roof of the buccal cavity. The pretrematic and posttrematic branches innervate the posterior and anterior halves, respectively, of the gill arches bordering a gill slit. Each branch splits into an internal and an external part. The internal bundle innervates the buccal side of the gill arch, including the gill rakers. The external bundle terminates in the gill filaments. The epibranchial motor branch, a small nerve bundle containing only motor fibers, circumvents the ganglion and anastomoses distally with the posttrematic branch. The detailed course and branching patterns of these branches are described.     

Associations between membranes and microtubules during mitosis and cytokinesis in caulonema tip cells of the mossFunaria hygrometrica

Summary The interphase nucleus in theFunaria caulonema tip cells is associated with many non-cortical microtubules (Mts). In prophase, the cortical Mts disappear in the nuclear region; in contrast to moss leaflets, a preprophase band of Mts is not formed in the caulonema. The Mts of the early spindle are associated with the fragments of the nuclear envelope. Remnants of the nucleolus remain in the form of granular bodies till interphase. The metaphase chromosomes have distinct kinetochores; the kinetochore Mts are intermingled with non-kinetochore Mts running closely along the chromatin. Each kinetochore is associated with an ER cisterna. ER cisternae also accompany the spindle fibers in metaphase and anaphase. In telophase, Golgi vesicles accumulate in the periphery of the developing cell plate where no Mts are found. The reorientation of the cell plate into an oblique position can be inhibited by colchicine. It is concluded that the ER participates in controlling the Mt system, perhaps via calcium ions (membrane-bound calcium ions have been visualized by staining with chlorotetracycline) but that, on the other hand, the Mt system also influences the distribution of the ER. The occurrence and function of the preprophase band of Mts is discussed.     

Phytochrome-mediated branch formation in protonemata of the mossCeratodon purpureus

We have analyzed light induction of side-branch formation and chloroplast re-arrangement in protonemata of the mossCeratodon purpureus. After 12 hr of dark adaptation, the rate of branch formation was as low as 5%. A red light treatment induced formation of side branches up to 75% of the dark-adapted protonema. The frequency of light induced branch formation differed between cells of different ages, the highest frequency being found in the 5th cell, the most distal cell studied from the apex. We examined the effect of polarized light given parallel to the direction of filament growth. The position of branching within the cell depended on the vibration plane of polarized red light. Branch formation was highest when the electric vector of polarized light vibrates parallel to the cell surface and is fluence rate dependent. The positional effect of polarized red light could be nullified to some extent by simultaneous irradiation with polarized far-red light. An aphototropic mutant,ptr116, shows characteristics of deficiency in biosynthesis of the phytochrome chromophore and exhibits no red-light induced branch formation. Biliverdin, a precursor of the phytochrome chromophore, rescued the red-light induced branching when added to the medium, supporting the conclusion that phytochrome acts as photoreceptor for red light induced branch formation. The light effect on chloroplast re-arrangement was also analyzed in this study. We found that polarized blue light induced chloroplast re-arrangement in wild-type cells, whereas polarized red light was inactive. This result suggests that chloroplast re-arrangement is only controlled by a blue light photoreceptor, not by phytochrome inCeratodon.