Nuclear behavior during branch formation in a centrifugedAdiantum protonema and the nuclear polarity

A new branch was induced on the side wall of fern protonema by cell centrifugation and subsequent polarized red light irradiation after the induction of cell division under white light. Nuclear behavior during the branch formation was analyzed. Immediately after cell division, the two daughter nuclei moved away from the division site in both red and dark conditions. Under continuous irradiation with polarized red light, cell swelling occurred as an early step of branching near the cell dividing wall, even though the nucleus was localized far from the branching site at the beginning of the swelling. After a new branch started to grow, the nucleus returned to the branching site and moved into the new branch from its basipetal end. When a protonema incubated in the dark was centrifuged again acropetally or basipetally just before the irradiation of polarized red light, the rate of apical growth or branch formation was increased, respectively. Moreover, growth of a branched protonema was altered from its former apex or from the branch again by dislocating the nucleus acropetally or basipetally by centrifugation, respectively. These facts suggest that the nucleus has no polarity physiologically, i.e. head and tail, namely either end of the spindle-shaped nucleus can be the nuclear front in a tip-growing protonema.     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.     

Die Wechselwirkung von Hellrot,Dunkelrot und Blaulicht bei der Photomorphogenese von Farngametophyten [Dryopteris filix-mas (L.) Schott]

Summary In Fig. 1 we have reproduced the action spectrum of photomorphogenesis in fern gametophytes (Dryopteris filix-mas (L.) Schott). The morphogenetic index L/W is shown as a function of wavelength (L=length, W=maximal width of the protonema). In experiments in which simultaneous irradiation with red and far-red was applied it has been shown (Fig. 2) that the effect of red light (lowering of the L/W-index) can be nullified by a simultaneous application of a suitable quantum flux density of far-red light. This fact means that the effects of red and far-red light on morphogenesis as measured by the L/W-index (Fig. 1) can be attributed exclusively to phytochrome.The strong morphogenetic effect of short wavelenth visible (=blue) light (strong lowering of the L/W-index) cannot be influenced by simultaneously applied far-red light (Fig. 4), whereas red light cancels the effect of blue light to a certain extent as measured by the L/W-index (Fig. 5). It has been concluded that the effect of blue light is due to a photoreceptor other than phytochrome, probably a flavoprotein. The antagonism between blue and red can be understood if we assume that the phytochrome-mediated growth at the tip of the apical cell of the protonema (e.g. Etzold, 1965) is fully promoted by P₇₃₀ only at a high relative concentration of P₇₃₀. The low relative concentration of P₇₃₀ under far-red light is too low to counteract significantly the blue light dependent response. Blue light initiates isodiametric growth of the apical cell instead of tip growth (Mohr, 1965). Under far-red light (a low level of P₇₃₀) growth of the apical cell seems to be restricted to the extreme tip of the apical cell. Slender protonemas with a high L/W-index are the result. Under red light (a high level of P₇₃₀) the growing zone of the apical cell is somewhat broader. As a consequence the protonemas are broader and the L/W-index is lowered.     

Changes in microtubule and microfibril arrangement during polarotropism inAdiantum protonemata

Polarotropism was induced inAdiantum (fern) protonemata grown under polarized red light by turning the electrical vector 45 or 70 degrees. One hour after the light treatment, tropic responses became apparent in many cells as a slight distortion of the apical dome. Changes in the position of the circumferentially-arranged cortical microtubule band (Mt-band) (Murataet al., 1987) and the arrangement of microfibrils around the subapical part of protonemata were investigated in relation to the polarotropic responses. Twenty minutes after turning the electrical vector, preceding the morphological change of cell shape, the Mt-band began to change its orientation from perpendicular to oblique to the initial growing axis. After 30 min, the Mt-band changed its orientation further under 45 degrees polarized light, but under light rotated 70 degrees, it began to disappear. In phototropic responses induced by local irradiation of a side of the subapical part of a protonema with a non-polarized red microbeam, the Mt-band on the irradiated side disappeared or became faint within 20 min, but neither disappearance nor a change of orientation of Mts occurred on the non-irradiated side. One hour after turning the electrical vector 45 degrees, in half of the cells tested, the innermost layer of microfibrils in the subapical part of the protonema changed its orientation from perpendicular to oblique to the growing axis, corresponding to the changes in the orientation of the Mt-band. After 2 hr, those changes were obvious in all cells examined. The same basic results on the orientation of microfibrils were obtained with protonemata cultured for 2 hr under 70 degrees polarized light. The role of the Mt-band in tropic responses is discussed.     

Intracellular Photoreceptive Site for Polarotropism in Protonema of the Fern Adiantum capillus-veneris L.

Polarotropic response was induced by short-term irradiationwith polarized red light in single-celled protonemata of thefern Adiantum capillus-veneris L. that had been grown apicallyunder red light for 6 days then for 15 hr in the dark. Sequentialobservation of the apical growth with a time-lapse video systemshowed that the direction of apical growth changed within 30min after the brief irradiation. Microbeam irradiation withpolarized red light of the subapical, dark-grown flank of theapical, 5–15 µm region of the protonema inducedthe polarotropic response most effectively. When both sidesof the flank were irradiated simultaneously with different fluencesof polarized red light with the same vibrating plane of 45°with protonemal axis, polarotropism took place normally, ifthe fluence ratio, B/A (B: fluence given to the side towardwhich the protonema should bend in polarotropism, A: fluencegiven to the other side) was not less than one-half. But, ifthe ratio became less than that, the protonemata no longer showedpolarotropism, they grew toward the side of higher fluence dependingon the difference in fluences between both sides. (Received August 1, 1981; Accepted September 29, 1981)     

The Position of Bud Differentiation on Protonema of the Moss, Physcomitrium sphaericum

The position of the gametophytic bud was examined in relationto the development of protonema in the moss, Physcomitrium sphaericum. Positions of protrusion formation, of the development of protrusionsinto lateral filaments, and of the differentiation of protrusionsinto buds are restricted within the narrow regions of the filaments.The number of cells from the apical cell of the filament tothese positions are constant in any size filament. The growth pattern of the protonema is shown as follow. As afilament grows one-dimensionally through divisions of the apicalcell, new protrusions are produced successively on the 5th cellfrom the apical cell or on its vicinity. The cells which intervenebetween the apical cell and this protrusion increase in numberas the apical cell divides. When this protrusion is positionedat the 8th or 9th cell from the apex, it differentiates intoa bud or a lateral filament. This growth pattern is common toboth the main and lateral filaments. Buds are differentiated not only on caulonema cells in the mainand lateral filament, but also on chloronema cells at the baseof the lateral filaments. (Received December 14, 1981; Accepted April 24, 1982)     

Photocontrol of the orientation of cell division inAdiantum

The rate of transition from one- to two-dimensional growth of fernAdiantum gametophytes under white light depends on the age of gametophyte cultured under red light. When gametophytes were cultured for longer period under red light, the rate of transition decreased and the number of abnormal gametophytes increased. Although the first step of the transition was the first longitudinal cell division following the two transverse ones (Wada and Furuya, 1970), the time-lapse-video study revealed that the apical cell of protonemata became flattened in the plane perpendicular to the incident ray of white light before the first longitudinal cell division. Analytical study of growing part of the apical cell with grains of activated charcoal as markers revealed that the apical cell flattening occurred evenly throughout the equatorial circumference of the cell even in the shaded side of the protonemata as well as in the side irradiated with white light.     

The distribution of plasmodesmata and its relationship to morphogenesis in fern gametophytes

Fern (Onoclea sensibilis) gametophytes when grown in the dark form a linear file of cells (one-dimensional) called a protonema. In the light two-dimensional growth occurs which results in a heart-shaped prothallus one cell thick. The objective of this paper is to relate the most common pattern of cell division observed in developing gametophytes to the formation of the plasmodesmatal network. Since the prothalli are only two dimensional, we can easily determine from thin sections the total number and the density (number per unit surface area) of plasmodesmata at each developmental stage. As the prothallus grows the number of plasmodesmata increases 50-fold in the apical or meristematic cell. This number eventually reaches a plateau even though the density continues to increase with each new cell division. What is particularly striking is that both the number and density of plasmodesmata between adjacent cells is precisely determined. Furthermore, the pattern of plasmodesmata distribution is predictable so that (1) we can identify the apical meristematic cells by their plasmodesmata number, or density, as well as by their size, shape and location, (2) we can predict, again from plasmodesmata number, the location of a future wall of the apical cell prior to its actual formation, (3) we can show that the density of plasmodesmata in the triangular apical cell of the prothallus (14 plasmodesmata microns-2) is comparable to those reported for secretory glands which are known to have high rates of plasmodesmatal transport and (4) we can show that once the plasmodesmata have been formed during division, no subsequent change in the number of plasmodesmata occurs following cell plate formation.     

Action spectra for polarotropism and phototropism in protonemata of the fern Adiantum capillus-veneris

The action spectrum for polarotropism was determined, using the Okazaki large spectrograph, by brief irradiation with light between 260 nm and 850 nm in single-celled protonemata of the fern Adiantum capillus-veneris L., which had been cultured for 6 days in red light and then in the dark for 15 h. The action spectrum had a peak at around 680 nm. This effect was nullified by subsequent irradiaton with far-red light, and typical red/far-red reversibility was observed, indicating the involvement of phytochrome. Polarized ultraviolet or blue light had no effect on the direction of apical growth. The action spectrum for phototropism was also determined in the red light region by means of brief microbeam irradiation of a flank of the subapical region of the protonema. This spectrum showed a peak at 662 nm which was consistent with the absorption peak of phytochrome, but not with the peak of the action spectrum for polarotropism.     

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.     

Electrical Changes in the Apical Cell of the Fern Gametophyte during Irradiation with Photomorphogenetically Active Light

Electrophysiological procedures were used to evaluate cellular responses of fern (Onoclea sensibilis L.) gametophytes to photomorphogenetically active light. Red, far red, and blue light caused rapid changes in the membrane potential of the apical cell of the gametophyte filament; other cells in the filament were not similarly responsive. Measurements made with one electrode in the apical cell revealed that the membrane potential depolarized in red light and repolarized in far red light. Irradiation with blue light caused a hyperpolarization, the rapidity of which was dependent on a red light pretreatment. More refined measurements with one electrode in the tip of the apical cell and another in the base of the cell showed that both red and blue light treatments cause the apical cell to behave as a dipole. Because of the profound, long-term morphological changes that follow light irradiation in this organism, it was hoped to use it to elucidate the role that electrical parameters play in determining subsequent developmental events.     

AN ULTRASTRUCTURAL STUDY OF FERN GAMETOPHYTES DURING ONE- TO TWO-DIMENSIONAL DEVELOPMENT

During early stages in the transition from 1- to 2-dimensional gametophyte development the change from filamentous to bulbous apical cell is not accompanied by major changes in the nature or number of cytoplasmic components of the cell. However, chloroplasts in apical cells of plants grown in red light are larger than those from cells of plants grown in blue light. In addition, the orientation of cytoplasmic microtubules is different in apical cells of plants from red and blue light. This change in orientation may be causally related to the change in apical cell form during 1- to 2-dimensional growth.     

Intracellular Localization and Dichroic Orientation of Phytochrome in Plasma Membrane and/or Ectoplasm of a Centrifuged Protonema of Fern Adiantum capillus-veneris L.

Protonemata of the fern Adiantum capillus-veneris grown undercontinuous red light for 6 days were kept in darkness for 15h and subsequently centrifuged 3 times in different directions,so that oil droplets and other cytoplasm were removed from theapical region of the protonemata. Electron micrographs clearlydemonstrated that cell wall, plasma membrane, ectoplasm andmicrotubules remained in the apical and subapical regions afterthe centrifugal treatments. A brief local exposure of the flankof the subapical region of the centrifuged protonemata to amicrobeam of red light effectively induced a phototropic responsetoward the irradiated side, suggesting that phytochrome is locatedin the ectoplasm and/or plasma membrane. When the flank of thecentrifuged protonema was irradiated with linearly polarizedred or far-red light, red light with an electrical vector parallelto the cell surface was more effective than that perpendicularto the cell surface. The direction of the electrical vectorof far-red light for reversion of the preirradiated red lighteffect, however, was opposite. These results suggest that differentdichroic orientations of P_R and P_FR exist in the plasma membraneor ectoplasm. (Received May 26, 1983; Accepted September 1, 1983)     

DETERMINATION OF RHIZOID ORIENTATION BY LIGHT AND DARKNESS IN GERMINATING SPORES OF ONOCLEA SENSIBILIS

When spores of the fern, Onoclea sensibilis L., are allowed to germinate in darkness, the rhizoid and the protonema are positioned at close to a right angle. If the spores are exposed initially to light and allowed to germinate, the rhizoid and protonema are positioned nearly axially, at opposite ends of the spore. The greater the duration and intensity of the initial illumination, the greater the tendency towards axial arrangement. All colors of light are active to some degree, and the effects are intensity-dependent. The response occurs in a uniform light field and is not dependent on a directional stimulus; the phenomenon reflects the relative arrangement of one part of the gametophyte to another part but not the orientation of growth with respect to an external stimulus. Direct tests show that neither the relative rhizoid orientation nor initial polarity of germination are affected by unilateral white light or polarized red light; the subsequent growth of the protonema, however, is oriented perpendicular to the plane of light polarization. The effects of light in determining the positional relationship between rhizoid and protonema are interpreted in terms of a hypothesis proposing light-induced changes in the structure and mechanical properties of the spore wall.     

Response of excised Avena coleoptile segments to red and far-red light

Summary In seeking a simple, red light-promoted straight growth test in which phytochrome assays may be conducted without interference by protochlorophyll, the response of excised Avena coleoptile segments to red and far-red light was re-examined. The elongation of apical (non-decapitated) segments is promoted by a brief exposure to red light, and this effect is almost completely nullified by an immediately subsequent exposure to far-red light. Although growth promotion by red light occurs in distilled water alone, the effect is greater on a medium consisting of 0.02 M phosphate buffer, pH 6.2 to 6.4, with 1 to 2% sucrose. Over the pH range 4.5 to 7.4, dark-growth decreases with increasing pH, but the absolute increment brought about by red light is nearly constant. Elongation appears to be entirely the result of increased cell size.Contrary to previous reports, similar results can be obtained with subapical (decapitated) coleoptile segments, although the absolute magnitude of the response is reduced.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commision.     

Daylength and development in four species of Ceramiaceae (Rhodophyta)

Developmental patterns in four species of Ceramiaceae were determined using excised thallus apices grown under a range of light periods. Models of thallus development and organization based on these patterns are presented. Increased rates of apical cell division, greater growth of apical fragments and increased average cell size were found with increasing number of hours light per day between 8–16 and 16–8 h. No aspect of growth investigated was associated with photoperiodic phenomena, and growth occuring during the light break (8-7.5-1-7.5 h) was intermediate between that in 8–16 and 12–12 h. Three patterns of cell elongation were found in the four species in which (1) cell age, (2) cell age and position and (3) cell age, cell position and light period determined cell length at different axial cell positions. Elongation was followed within cells, along axes ofAntithamnion spirographidis for plants grown under different day lengths. Three regions of development were found along main axes: (1) an apical region in which basipetal expansion was greater than acropetal expansion. (2) a zone of stability with equal elongation in apical and basal growth region of cells, and (3) a basal region with greater acropetal expansion. With increasing daylength, the zone of stability was extended to greater ranges of cell length.     

A quantitative and dynamic model for plant stem cell regulation

Plants maintain pools of totipotent stem cells throughout their entire life. These stem cells are embedded within specialized tissues called meristems, which form the growing points of the organism. The shoot apical meristem of the reference plant Arabidopsis thaliana is subdivided into several distinct domains, which execute diverse biological functions, such as tissue organization, cell-proliferation and differentiation. The number of cells required for growth and organ formation changes over the course of a plants life, while the structure of the meristem remains remarkably constant. Thus, regulatory systems must be in place, which allow for an adaptation of cell proliferation within the shoot apical meristem, while maintaining the organization at the tissue level. To advance our understanding of this dynamic tissue behavior, we measured domain sizes as well as cell division rates of the shoot apical meristem under various environmental conditions, which cause adaptations in meristem size. Based on our results we developed a mathematical model to explain the observed changes by a cell pool size dependent regulation of cell proliferation and differentiation, which is able to correctly predict CLV3 and WUS over-expression phenotypes. While the model shows stem cell homeostasis under constant growth conditions, it predicts a variation in stem cell number under changing conditions. Consistent with our experimental data this behavior is correlated with variations in cell proliferation. Therefore, we investigate different signaling mechanisms, which could stabilize stem cell number despite variations in cell proliferation. Our results shed light onto the dynamic constraints of stem cell pool maintenance in the shoot apical meristem of Arabidopsis in different environmental conditions and developmental states.     

The Relationship between the Kinetics of Ribonucleic Acid Accumulation and the Morphological Development of the Fern Gametophyte, Dryopteris borreri

Fern gametophytes were grown under blue light with and without the addition of 5-fluorouracil or 8-azaguanine, and under red light. Nucleic acids were extracted by either the detergent-chloroform or the detergent-diethylpyrocarbonate method and analyzed by polyacrylamide gel electrophoresis. No significant differences in the relative distribution of the stable RNA components accompanied the transition to biplanar growth. The RNA content per average cell decreased with growth and also varied between the cultural conditions, yet it was independent of the pattern of morphological development. The falling RNA content per average cell resulted from a progressive reduction of the RNA content of the apical cell, as determined histochemically. Since filamentous growth occurred by division of this apical cell, the rate of cell division was independent of the RNA content of the dividing cell.     

Growth of Peach Plants under Different Filtered Sunlight Conditions

Young peach plants (Prunus persica) were grown outdoors under different colored filters, to examine the effect of light quality on plant behavior. It was found that under blue light growth rate, leaf size and number, rate of spring bud opening and secondary branching were very similar to control plants grown under neutral shade. Blue + far-red light showed an overall strong inhibitory effect on all these characteristics. Red + far-red light produced the strongest growth activity with best results in growth rate and leaf size and number. The phytochrome pigment system was suggested to be the only pigment regulating growth under high light intensities. Blue and blue + far-red light acted antagonistically on apical dominance features of the tree. The former produced a wider tree with nearly horizontal shoots, while the latter produced a more erect narrow tree.