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.     

Irradiance-dependent regulation of gravitropism by red light in protonemata of the moss Ceratodon purpureus

Apical cells of protonemata of the moss Ceratodon purpureus (Hedw.) Brid. are negatively gravitropic in the dark and positively phototropic in red light. Various fluence rates of unilateral red light were tested to determine whether both tropisms operate simultaneously. At irradiances ≥140 nmol m⁻² s⁻¹ no gravitropism could be detected and phototropism predominated, despite the presence of amyloplast sedimentation. Gravitropism occurred at irradiances lower than 140 nmol m⁻² s⁻¹ with most cells oriented above the horizontal but not upright. At these low fluence rates, phototropism was indistinct at 1 g but apparent in microgravity, indicating that gravitropism and phototropism compete at 1 g. The frequency of protonemata that were negatively phototropic varied with the fluence rate and the duration of illumination, as well as with the position of the apical cell before illumination. These data show that the fluence rate of red light regulates whether gravitropism is allowed or completely repressed, and that it influences the polarity of phototropism and the extent to which apical cells are aligned in the light path. Received: 19 January 1999 / Accepted: 19 March 1999     

蕨类植物配子体发育与生理生态研究进展

蕨类植物配子体为单倍体,结构简单,独立于孢子体生活,在研究其对环境的响应以及揭示其机理上都具有独特的优势。该研究从我国和国际两个分支出发,梳理了近年来全球范围内相关的文献资料,透视了蕨类配子体的发育和生理生态前沿科学和研究动态。在发育部分以研究进展为主要内容,国内研究以传统植物蕨类植物的配子体形态和发育的观察为主,而国外学者更关注于新技术和新方法在传统学科中的运用,如X光透射技术和流式细胞术。生理生态部分分为光合与呼吸作用、土壤逆境的响应、气候变化的响应以及对化感物质的响应4个板块。在光合作用的研究中,发现蕨类配子体会在光强的变化下产生自我保护机制,碳水化合物和脂质是配子体能量代谢中的重要指标。在土壤逆境的响应研究中,对砷有超富集作用的蜈蚣草配子体和耐高盐的铁角蕨配子体是配子体研究中较为突出的材料。荷兰地区广泛存在的耳蕨属蕨类,哥斯达黎加热带雨林的20种蕨类植物及水生蕨类槐叶萍,成为了证明配子体成活率和温度之间重要关系的实验例证。在化感作用研究中,主要通过紫茎泽兰根、茎和叶水提液对扇蕨等4种蕨类配子体的作用,证明了入侵植物对于蕨类植物配子体生长发育具有危害作用。在美国佛罗里达的一类爬树蕨也发现了同样的入侵植物现象。此外,还对几个新兴技术在配子体研究的前景进行了展望,并对已有技术进行了描述。该研究以多个角度介绍了国内外配子体的研究进展,希望有助于促进我国学者对该领域的深入研究。     

Regeneration of drought-stressed gametophytes of the epiphytic fern, Pyrrosia pilosellodes (L.) Price

The epiphytic habitat represents a highly dynamic environment, and water deficit is one of the common factors that affects growth and development of epiphytes. Gametophytes of the epiphytic fern, Pyrrosia piloselloides (L.) Price, were able to tolerate up to 50 days of drought. Upon rehydration, cells that recovered from water stress were capable of forming new gametophytes. The ability of gametophytes to recover from desiccation plays an important role in the survival and growth of the fern species under natural conditions. Received: 20 May 1998 / Revision received: 5 August 1998 / Accepted: 21 August 1998     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. I. Asynchronous mitosis of two nuclei in a single cell

Coordination of karyokinesis of two nuclei in individual filamentous binucleate cells of the fern,Adiantum capillus-veneris was investigated. To induce binucleate cells, the protonemata were treated with caffeine, which is known as an inhibitor of plant cytokinesis, during the first synchronous division of cells that was induced by blue light (BL). The next synchronous division of cells in the resultant binucleate cells was analysed. In most cases, the two nuclei were associated with each other and were located in the apical region of the long protonemal cells (approximately 400–600 μm in length, 20 μm in width). In some cells, one nucleus was located in the apical region and the other was located in the middle of the cylinderical region. In such cells, karyokinesis of the apical nucleus preceded that of the basal nucleus, even though karyokinesis of associated nuclei progressed synchronously. Mitotic binucleate cells were centrifuged in order to gather two dissociated heterophasic nuclei. Progression of karyokinesis in the re-associated nuclei became coordinated within 1 h in most cells. These results suggest that mitosis-regulating factor(s) may diffuse to only limited distances inAdiantum protonemata.     

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)     

trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae)

Background and Aims

The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given.

Methods

A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST.

Key Results trnL-F

earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts.

Conclusions

This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Drynaria fortunei</Emphasis>, a fern species source of Chinese medicine “Gu-Sui-Bu”

This study reports spore germination, early gametophyte development and change in the reproductive phase of Drynaria fortunei, a medicinal fern, in response to changes in pH and light spectra. Germination of D. fortunei spores occurred on a wide range of pH from 3.7 to 9.7. The highest germination (63.3%) occurred on ½ strength Murashige and Skoog basal medium supplemented with 2% sucrose at pH 7.7 under white light condition. Among the different light spectra tested, red, far-red, blue, and white light resulted in 71.3, 42.3, 52.7, and 71.0% spore germination, respectively. There were no morphological differences among gametophytes grown under white and blue light. Elongated or filamentous but multiseriate gametophytes developed under red light, whereas under far-red light gametophytes grew as uniseriate filaments consisting of mostly elongated cells. Different light spectra influenced development of antheridia and archegonia in the gametophytes. Gametophytes gave rise to new gametophytes and developed antheridia and archegonia after they were transferred to culture flasks. After these gametophytes were transferred to plastic tray cells with potting mix of tree fern trunk fiber mix (TFTF mix) and peatmoss the highest number of sporophytes was found. Sporophytes grown in pots developed rhizomes.     

PHOTOMORPHOGENESIS OF THE GAMETOPHYTES OF LYGODIUM JAPONICUM

Protonemata of Lygodium japonicum turn biplanar in both red and blue light regimes and remain filamentous in far-red light. Biplanar gametophytes formed in red light are longer than broad with long, rectangular cells, whereas in blue light they appear broader than long with short, isodiametric cells. Transfer of protonemata of all ages from far-red regime to red or blue light induces a morphological type of growth characteristic of the new light regime. However, only relatively young biplanar forms transferred from red or blue light are able to resume filamentous type of growth in a subsequent regime of far-red light.     

Ethylene is a modulator of gibberellic acid-induced antheridiogenesis in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.     

Bending growth in fern gametophyte protonema

Filamentous young gametophytes of the fern, Pteridium aquilinum,exhibit a positive phototropism in unilateral light. The displacementof artificial markers demonstrated that this new direction ofgrowth was due to the repositioning of the apical growth tiprather than an increased growth on the dark side. (Received December 16, 1974; )     

亚热带10种蕨类植物光响应模型拟合及光合特性研究

通过合适的光响应模型来准确计算植物光合参数是研究植物光合特性的重要手段。为深入了解亚热带蕨类植物的光合特性,采用直角双曲线模型、非直角双曲线模型、指数模型和直角双曲线修正模型等4种常用光响应模型分别对10种亚热带蕨类植物进行光响应曲线拟合。通过分析决定系数(R~2)、均方根误差(RMSE)、平均绝对误差(MAE)、光饱和点(LSP)、光补偿点(LCP)、最大净光合速率(P_nmax)、暗呼吸速率(R_d)和表观量子效率(AQE)等9种参数,探讨亚热带蕨类植物的光响应模型拟合效果及其光合特性。结果表明：直角双曲线修正模型和非直角双曲线模型对本研究10种亚热带蕨类植物的拟合效果较好,其中直角双曲线修正模型的LSP拟合效果最好,非直角双曲线模型的拟合精度最高。光合参数的聚类分析和TOPSIS(Technique for Order Preference by Similarity to an Ideal Solution)综合评价法表明,10种亚热带蕨类植物的光合特性既具有相似性又存在物种特异性。黑鳞耳蕨(Polystichum makinoi(Tag...     

Red light enhancement of the phototropic response of etiolated pea stems

In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using ¹⁴C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of ³H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment.     

The effects of methionine and cobalt ions on sex expression and development of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

One of the prime precursor for ethylene synthesis — L-methionine and the inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) — Co²⁺-were tested for their effects on sex expression and development of Anemia phyllitidis fern gametophytes. Five concentrations of both chemicals (0, 10, 25, 50, 100 μM) were analysed with reference to antheridia and archegonia formation, number and size of cells as well as thalli length using the three-zone model of gametophyte structure. Both substances, however at different concentrations, enhanced the number of GA₃-induced antheridia and similarly stimulated the cell number and inhibited thalli length. Both of them at 100 μM concentrations without GA₃ induced meristematic area formation while methionine also induced archegonia in the apical parts of gametophytes. These findings correspond with the previous observations concerning the important role of ethylene synthesis precursor (ACC) in controlling gibberellic acid-induced male sex expression in ferns and broaden the knowledge about the mechanisms of fern gametophyte development.     

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.     

GAMETOPHYTES AND YOUNG SPOROPHYTES OF OPHIOGLOSSUM CROTALOPHOROIDES WALT.

The lens-shaped, dorsiventral gametophytes of Ophioglossum crotalophoroides are superficially different from the cylindrical gametophytes of other species of Ophioglossum. However, they have the same features as other Ophioglossum gametophytes (fundamentally axial organization, radially symmetrical apical meristem, radial distribution of gametangia) except that the cylindrical axis is reduced in length. Young sporophyte development is unique in the genus: all primary organs clearly arise from the embryo and develop simultaneously. The length of the life cycle of O. crotalophoroicles is considerably shorter than that of some other species in the Ophioglossaceae. A timetable for gametophyte and young sporophyte development is postulated. Spores germinate soon after they are released in the spring, and mature gametophytes develop by the next growing season. Fertilization occurs approximately one year after spore dispersal, and after two years, the photosynthetic first leaf of the young sporophyte emerges.     

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.