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.     

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.     

Effects of colchicine and amiprophos-methyl on microfibril arrangement and cell shape inAdiantum protonemal cells

Summary 5 mM colchicine and 1 g/ml amiprophos-methyl, known antimicrotubule agents, were applied to fernAdiantum protonemata under red light. Both drugs caused microtubule disruption and subsequent apical swelling of protonemal cells after certain lag periods. While the lag periods for the onset of microtubule disruption after application of the two drugs were different (within 15 minutes in amiprophos-methyl, 1 hour in colchicine), the lag periods of apical swelling after microtubule disruption were nearly the same (approx. 70 minutes). The results suggest that the apical swelling is a consequence of microtubule disruption.In cells examined 1 hour after microtubule disruption by either drug, the microfibril arrangement of the innermost layer of the cell wall was random at the tip, transverse in the subapical region, and roughly longitudinal in the cylindrical region. This pattern of microfibrils was similar to that of untreated cells in which the microtubules show a similar arrangement (Murata and Wada 1989). Surprisingly, even after approx. 4 hours of microtubule disruption, when apical swelling had occurred in most cells, the pattern of microfibril deposition was not altered. The role of microtubules in oriented microfibril deposition and the mechanism of control of cell shape are discussed.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - MT(s) microtubule(s) - PBS phosphate buffered saline     

Circular arrangement of cortical microtubules around the subapical part of a tip-growing fern protonema

Summary Cortical microtubule arrays in tip-growing protonemal and rhizoid cells of the fernAdiantum gametophytes were observed by immunofluorescence microscopy. A circular arrangement of cortical microtubules was demonstrated around the subapical part of protonemal cells growing under red light conditions. However, such an arrangement was not found in growing rhizoids either by immunofluorescence microscopy or by electron microscopy. The different patterns of microtubule arrays around the apices of tip-growing protonemal and rhizoid cells suggest the possible existence of different mechanisms in regulating the cell diameter in the two types of cylindrical cell.     

A model system to study the effect of SO2 on plant cells. III. Effects of sulfite on the ultrastructure of fern protonemal cells

The mechanism of the toxic effects on plant cells of sulfite, a product of the air pollutant sulfur dioxide, is not well understood. Therefore, changes in the fine structure and organization of microtubules and microfibrils induced by sulfite were studied by electron and light microscopy in the protonemata of the fernAdiantum capillusveneris L. Under red-light conditions, growing protonemata fumigated with 0.05 or 0.1 μ1/1 SO₂ for 1 to 4 days showed abnormalities, such as apical swelling, and they sometimes burst at the apex. The incidence of abnormalities seemed to be correlated with the concentration of the sulfite dissolved in the culture medium. At an appropriate concentration (3.3–6.6. mM) of sulfite (applied as K₂SO₃), cell swelling at the apical region of protonema was also induced. When the concentration of sulfite was as high as 6.6 mM, more than 60% of protonemata burst at the tip. During the apical swelling, no distinct changes were observed in the fine structure of organelles, such as the chloroplasts, mitochondria, microbodies, Golgi bodies and nucleus. However, the arrangement of cortical microtubules and that of the innermost layer of microfibrils around the subapical region of protonemata were changed from transverse to the cell axis (i.e., circular) to random and the cell wall was thickened. These observations suggest that sulfite may influence the mechanisms that maintain the transverse orientation of microtubules in the subapical region of a protonema and that the resultant random arrangement of microtubules induces the random arrangement of microfibrils and leads to apical swelling.     

Chloroplasts do not have a polarity for light-induced accumulation movement

Chloroplast photorelocation movement in green plants is generally mediated by blue light. However, in cryptogam plants, including ferns, mosses, and algae, both red light and blue light are effective. Although the photoreceptors required for this phenomenon have been identified, the mechanisms underlying this movement response are not yet known. In order to analyze this response in more detail, chloroplast movement was induced in dark-adapted Adiantum capillus-veneris gametophyte cells by partial cell irradiation with a microbeam of red and/or blue light. In each case, chloroplasts were found to move toward the microbeam-irradiated area. A second microbeam was also applied to the cell at a separate location before the chloroplasts had reached the destination of the first microbeam. Under these conditions, chloroplasts were found to change their direction of movement without turning and move toward the second microbeam-irradiated area after a lag time of a few minutes. These findings indicate that chloroplasts can move in any direction and do not exhibit a polarity for chloroplast accumulation movement. This phenomenon was analyzed in detail in Adiantum and subsequently confirmed in Arabidopsis thaliana palisade cells. Interestingly, the lag time for direction change toward the second microbeam in Adiantum was longer in the red light than in the blue light. However, the reason for this discrepancy is not yet understood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Formation of novel endoplasmic and cortical microtubular arrays inAdiantum protonemal cells induced by blue light irradiation and acropetal centrifugation

Protonemal cells ofAdiantum capillus-veneris were grown under red light conditions over 6 days and exposed to blue light for 8 hr (and then dim green light for 1 hr for technical reasons), before they were centrifuged acropetally over at least 1 hr at 2,000×g. After this treatment, an arrangement of endoplasmic microtubules (MTs) that resembled the shape of a tadpole could be detected some distance below the nucleus in about 40% of the cells. The percentage of protonemata bearing this Mtstructure was dependent on centrifugation time as well as the time of blue light irradiation. The size of the structure was constant at any time of its existence. Additionally, a wide belt of transversally oriented cortical MTs in the upper part of the protonemata was detected in many cells after blue light irradiation and acropetal centrifugation. Its formation rate seemed to be also dependent on the period of blue light irradiation and centrifugation time. None of the endoplasmic and few of the cortical transverse MT patterns could be seen without blue light irradiation. A strict coincidence in the formation of both MT patterns was not seen. Further, a few tadpole-shaped MT arrays remained during mitosis, whereas the cortical transverse MT pattern was found in stages other than metaphase and anaphase.     

Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fernAdiantum

Orientational movement of chloroplasts was induced by a brief irradiation with red light (R) or blue light (B) in dark-adapted prothallial cells ofAdiantum, whose chloroplasts had gathered along the cell dividing wall (i.e., the anticlinal wall). When the whole dark-adapted prothallia were irradiated from a horizontal direction (i.e., from their lobes) with horizontally vibrating polarized R (H pol. R) for 10 or 3 min, the chloroplast left the anticlinal walls and spread over the prothallial surface (i.e., the periclinal walls) within 1–2 hr after the onset of irradiation, returning to the anticlinal wall (dark-position) within 10 hr. However, vertically vibrating polarized R (V pol. R) for 10 min did not induce the movement towards periclinal walls. The R effect was cancelled by non-polarized far-red light (FR) irradiation just after the R irradiation. Irradiation with H pol. B for 10 or 3 min but not with V pol. B could also induce a similar movement of chloroplasts, although the chloroplasts returned within 4 hr. The effect of H pol. B, however, was not cancelled by the subsequent FR irradiation. When a part of the dark-adapted cell at the prothallial surface was irradiated from above with a microbeam of R or B for 1 min, chloroplasts of the cell in the dark-position moved towards the irradiated locus in subsequent darkness. However, in the neighboring cells, orientational movement was not induced by either R or B microbeams. These results show that in dark-adapted prothallial cells, both brief irradiation with R and B can induce chloroplast photo-orientation and that the photoreceptors are phytochrome and blue light-absorbing pigment, respectively. It is also clear that effects of both R and B irradiation do not transfer to neighboring cells.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

Phytochrome-mediated polarotropism of Adiantum capillus-veneris L. protonemata as analyzed by microbeam irradiation with polarized light

Summary Perception of polarized light inducing phytochrome-mediated polarotropism in protonemata of the fern Adiantum capillus-veneris L. was analyzed using brief microbeam irradiation with polarized red (R) or far-red light (FR). The polarotropic response inducible by irradiation of the subapical 10–30-m part with polarized R vibrating parallel to the cell axis was nullified by subsequently giving R at the apical 0–2.5-m region. This inhibitory effect of R showed an action dichroism, that is, polarized R vibrating normal to the cell axis was effective but the parallel-vibrating R was not. On the other hand, FR irradiation of the extreme tip after irradiation of the whole cell with depolarized R effectively induced a tropic response. This FR effect also showed action dichroism, with parallel-vibrating polarized FR being more effective than FR vibrating normal to the cell axis. When the apical-dome region and the adjacent subapical 10–20-m region were sequentially irradiated with polarized R vibrating obliquely in different directions, polarotropism took place depending on the vibrating direction of the light given to the apical-dome region. Obliquely vibrating polarized FR given to the apical dome after irradiation of the whole cell with depolarized R also induced polarotropism. Thus, the difference in amount (or percent) of the far-redabsorbing form of phytochrome (Pfr) between the extreme tip and the subapical region appears to be crucial in regulating the direction of apical growth; the difference in Pfr level between the two sides of the protonemal apex may occur mainly at the apical dome. Furthermore, the transition moments of the red-absorbing form of phytochrome (Pr) and Pfr seem to be aligned parallel and normal, respectively, to the cell surface at the periphery of the apical hemisphere.Abbreviations FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

The changes of nuclear position and distribution of circumferentially aligned cortical microtubules during the progression of cell cycle inAdiantum protonemata

The intracellular positions of the nucleus and of cortical, circumferentially aligned microtubules (CCAM) in filamentous, single-celled protonemata ofAdiantum capillus-veneris were determined throughout the cell cycle in the dark. When apical growth continued at G₁ phase, the nucleus migrated keeping a constant distance from the tip. When the apical growth stopped at late S or G₂ phase, the nucleus stopped moving forward and then slightly moved backward to the site of cytokinesis. The CCAM were found only in the dome of protonemal tip when growing under continuous red light; they increased in number after dark incubation for 12 hr and then decreased after 20th hr in the dark. The CCAM were usually observed in the region between the nucleus and the tip at 28 hr in the dark. They were located around the nuclear region at pre-prophase and prophase, but then totally disappeared at metaphase and thereafter.     

Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

Reorganization of the cortical cytoskeleton in tip-growing fern protonemal cells during phytochrome-mediated phototropism and blue light-induced apical swelling

Summary Circular arrays of cortical microtubules (MTs) and microfilaments (MFs) are found in the subapical region of tip-growing protonemal cells of the fernAdiantum capillus-veneris. Reorganization of the two cytoskeletal structures during phytochrome-mediated phototropism and blue light-induced apical swelling was investigated by double-staining of MTs and MFs with rhodaminephalloidin and an indirect immunofluorescence method with tubulinspecific antibody. Before any growth responses were detectable, the MF and MT structures were reorganized according to similar patterns in both photoresponses, that is, oblique orientation and transient disappearance of the structures occurred during the phototropic response, and the disappearance of the structures occurred during apical swelling. The reorganization of MF structures clearly preceded that of the MT structures in the phototropic response. In the case of apical swelling, both types of circular array disappeared with an almost identical time course.These results provide evidence for the significant role of the circular organization of MFs as well as of MTs, in the light-induced growth responses of tip-growing fern protonemal cells. Possible roles of the circular array of MFs in the regulation of tip growth are discussed.Abbreviations DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - MF microfilament - MT microtubule - Rh-Phal rhodaminelabeled phalloidin     

Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

Plastic responses to light intensity and planting density in three Lamium species

In this survey plastic responses to light intensity and planting density were examined in three Lamium species (L. purpureum, L. album and L. maculatum). Low light intensity enhanced plant height, length and width of leaves, but reduced number of shoots and leaves, as well as root and shoot weights. Higher density resulted in smaller plants and leaves, but had significant effect on module number (shoots and leaves) only on older plants. The effect of light intensity on measured traits was greater than the effect of density, and consistent with predictions about plastic responses on light intensity variation. Generally, the three Lamium species differed in the magnitude but not in patterns of plasticity. However, associations of analyzed traits with fitness significantly differed among species as well as among light treatments.     

Freeze-fracture observations on the plasma membrane,the cell wall and the cuticle of growing protonemata of Adiantum capillus-veneris L.

Using freeze-fracture electron microscopy we have examined the morphology of the plasma membrane and the cell wall of single-celled protonemal filaments of the fern Adiantum capillus-veneris grown under continuous red light. The surface of the protonemal cell wall is completely covered by a multilayered, lipid-like coat, probably consisting of cuticular waxes. The rhizoid seems to lack this type of coat. The cell walls of the protonemata contain 8-nm thick, randomly oriented fibrils. In rapidly growing protonemata the P-face of the plasma membrane contains both randomly distributed particles and distinct particle rosettes. The rosettes consist of six 8–9-nm-wide particles in a ring-like configaration and have an outer diameter of 24 nm. They closely resemble the particle rosettes seen on the P-face of the plasma membrane of green algae and of higher plants, which recently have been implicated in the synthesis of cellulose fibrils. Within 20 m from the tip of the protonemata, and coinciding with the region of maximal cell-wall growth and expansion and thus cellulose-fibril synthesis, the greatest density of rosettes (20/m²) is observed. Beyond 20 m from the tip this number drops rapidly to near zero at 50 m. The rosettes have a tendency to form small, irregular clusters, but only very rarely are three or more rosettes found in a row or in a geometrical pattern. Our measurements of the size and the density of the randomly distributed plasma membrane particles indicate that the tip region must be specialized with respect to other plasma-membrane activities as well. Thus the tip region contains not only the highest density of randomly destributed intramembrane particles, but also particles of different sizes than those found elsewhere in the plasma membrane.     

Chlorophyll-proteins from maize seedlings grown under intermittent light conditions

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.Abbreviations Chl chlorophyll - CL continuous light - F fraction - HPLC high-performance liquid chromatography - IEF isoelectrofocussing - IL intermittent light - LHCII light-harvesting complex II - PAGE polyacrylamide-gel electrophoresis - Phe pheophytin - SDS sodium dodecyl sulfate This work was supported by the grant no. 4.7240.90 from the Italian Ministry of Agriculture and Forestry. We thank Drs. R. Barbato (Dipartimento di Biologia, Padua, Italy) and Olivier Vallon (Institut de Biologie Physico-Chimique, Paris, France) for their gifts of antibodies, Drs. R. Barbato and P. Dainese (Dipartimento di Biologia, Padua, Italy) for fruitful discussion and Prof. G. Gennari (Dipartimento di Chimica fisica, Padua, Italy) for his assistance in recording the excitation spectra. J.M. was supported by a Stipendium from the Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.     

Effects of blue and red light on unrolling of rice leaves

Unrolling of the second leaf of 8-day-old rice (Oryza sativa L.) seedlings was promoted by weak blue light (B), but not by red light (R). The effect of B was counteracted by irradiation with R just before or after the B. The counteracting effect of R was reversed by subsequent irradiation with far-red light but not by B, even if B was applied for 10 h. The B was effective when the region 0.5–2 cm from the tip of the leaf was irradiated. These results indicate that in rice photoreceptors for blue light located in the region 0.5–2 cm from the tip of the leaf play a key role in leaf unrolling and that a B-absorbing pigment and phytochrome participate in leaf unrolling in a closely related manner.Abbreviations B blue light - R red light - FR far-red light - W white light - D dark This work was presented at the Annual Meeting of the Japanese Society of Plant Physiologists on April 4, 1978, in Hiroshima