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     

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

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

Amyloplasts as possible statoliths in gravitropic protonemata of the moss Ceratodon purpureus

The kinetics of gravitropism and of amyloplast sedimentation were studied in dark-grown protonemata of the moss Ceratodon purpureus (Hedw.) Brid. The protonemata grew straight up at a rate of 20–25 m·h^– in nutrient-supplemented agar. After they were oriented to the horizontal, upward curvature was first detected after 1–1.5 h and reached 84° by 24 h. The tip cells exhibited an amyloplast zonation, with a tip cluster of nonsedimenting amyloplasts, an amyloplast-free zone, and a zone with pronounced amyloplast sedimentation. This latter zone appears specialized more for lateral than for axial sedimentation since amyloplasts sediment to the lower wall in horizontal protonemata but do not fall to the basal wall in vertical protonemata. Amyloplast sedimentation started within 15 min of gravistimulation; this is within the 12–17-min presentation time. The data support the hypothesis that some amyloplasts function as statoliths in these cells.This work was supported by the National Aeronautics and Space Administration grant NAGW-780. We thank Professor E. Hartmann and J. Schwuchow for providing Ceratodon cultures, Dr. John Z. Kiss and Jeff Young for valuable discussions, and Professor Rainer Hertel (University of Freiburg, FRG) for bringing this material to our attention.     

Amyloplasts that sediment in protonemata of the moss Ceratodon purpureus are nonrandomly distributed in microgravity

Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates for g-induced drag while still allowing for regulated amyloplast sedimentation.     

Ultrastructural analysis of cell component distribution in the apical cell ofCeratodon protonemata

Summary A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the mossCeratodon. The first 35 m of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tipmost 5 m and evenly distributed throughout the remaining 30 m. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.Abbreviations CER cisternal endoplasmic reticulum - ER endoplasmic reticulum - N_d numerical density - SE standard error - S_v surface density - TEM transmission electron microscopy - TER tubular endoplasmic reticulum - V_v volume fraction     

Microfilament distribution in protonemata of the mossCeratodon

Summary Microfilaments were visualized in dark-grown protonemata of the mossCeratodon to assess their possible role in tip growth and gravitropism. The relative effectiveness of rhodamine phalloidin (with or without MBS) and of immunofluorescence (using the C4 antibody) was evaluated for actin localization in the same cell type. Using immunofluorescence, microfilaments were primarily in an axial orientation within the apical cell. However, a more complex network of microfilaments was observed using rhodamine phalloidin after MBS pretreatment, especially when viewed by confocal laser scanning microscopy. This method revealed a rich three dimensional network of fine microfilaments throughout the apical cell, including the extreme apex. Although there were numerous internal microfilaments, peripheral microfilaments were more abundant. No major redistribution of microfilaments was detected after gravistimulation. The combination of MBS, rhodamine phalloidin, and confocal laser scanning microscopy preserves and reveals microfilaments remarkably well and documents perhaps the most extensive F-actin network visualized to date in any tip-growing cell.Abbreviations BSA bovine serum albumin - CLSM confocal laser scanning microscopy - DIC differential interference contrast - DMSO dimethylsulfoxide - EGTA ethylene glycol bis-(-amino-ethylether) N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MEOH methanol - PBS phosphate buffered saline - PFA paraformaldehyde - PIPES piperazine-N,N-bis-2-ethanesulfonic acid - PMSF phenylmethyl sulfonyl fluoride - RP rhodamine phalloidin     

Time-lapse analysis of the circadian rhythms of conidiation and growth rate in neurospora

The filamentous fungus Neurospora crassa has frequently served as a model organism for the study of circadian rhythms through its ability to form conidial spores on a daily basis. This phenomenon leaves a spatial pattern of conidiation bands along a solid surface of agar after several days of growth. Using time-lapse video, the authors have quantified the rate of conidiation. They have found that conidia do not form at a specified lag time after the growth front is laid down, but rather the band region tends to simultaneously develop over a short time frame. This produces a sharp peak when the conidiation rate is plotted against time. In addition, the authors used time-lapse video to assay growth rate with greater accuracy than previously reported. It is usually assumed that Neurospora's rate of growth is constant, and this assumption of linear growth has been used extensively to determine period and phase of the conidiation circadian rhythm. The authors have confirmed an earlier report of nonlinear growth rate and have shown that the growth rate varies by a factor of about 2 with each circadian cycle. They have demonstrated that the errors in calculating times of conidiation peaks are maximally 1 to 2 h if linearity is assumed. The conidiation rate and growth rate rhythms are not apparent under conditions (using mutants or high or low temperatures) where the spatial banding rhythm is not observed. In light/dark entraining conditions, the conidiation rate and growth rate rhythms maintain the same phase relationship in different T-cycles. These data are consistent with the hypothesis that the growth rate rhythm is a consequence of the conidiation rate rhythm.     

Apical growth of protonemata inAdiantum capillus-veneris

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

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with the subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenk?rper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289–298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenk?rper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropism in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton. Received: 14 September 1997 / Accepted: 16 October 1997     

Microtubule distribution in gravitropic protonemata of the mossCeratodon

Summary Tip cells of dark-grown protonemata of the mossCeratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for >20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethylene glycolbis-(-amino-ethylether) N,N,N',N'-tetraacetic acid - FITC fluorescein isothiocyanate - GS gravitropic stimulus - MT microtubule - PIPES piperazine-N,N'-bis-2-ethanesulfonic acid     

Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis

Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininβ1 or lamininγ1 contrast with later dystrophic phenotypes in lamininα2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.     

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.     

Isolation of genes involved in gravitropism.

When upright growing wild type barley seedling are placed to a horizontal position they will respond to this gravistimulation by a curvature against the gravitational force. The mutant serpentina, however, shows gravitropic response only in an early stage of development. Older seedlings, gravistimulated 92 h after germination are agravitropic. They will grow horizontally. We try to identify genes involved in gravitropism by comparing gene expression of i) non-gravistimulated and gravistimulated wild type barley seedlings and of ii) gravistimulated wild type and serpentina.     

Sequence of key events in shoot gravitropism

It has recently been shown that asymmetric acid efflux is closely correlated with the gravitropic curvature of plant shoots and roots. The research reported here addresses whether auxin (IAA) redistribution in shoots is the cause or result of asymmetric acid efflux.

When abraded sunflower (Helianthus annuus cv Mammoth) hypocotyls are submerged in 20 millimolar neutral buffer, gravicurvature is greatly retarded relative to 0.2 millimolar controls. Nevertheless, in both buffer systems there is a similar redistribution of [³H]IAA toward the lower surface of gravistimulated sunflower hypocotyls. These results suggest that graviperception initiates IAA redistribution, which in turn results in auxin-induced asymmetric H⁺ efflux across the shoot. This interpretation is reinforced by data showing the effects of removal of the epidermal layers (peeling), osmotic shock, and morphactin treatment on gravicurvature and [³H]IAA redistribution. Peeling and osmotic shock inhibit gravicurvature but not redistribution. Morphactin inhibits both processes but does not inhibit hypocotyl straight growth.

     

Anisotropy of wave velocities in plants: gravitropism

New measurements of W-wave velocities in plants suggest that the previously observed velocity of 96 cm/s in plants is not unique but that there are several higher velocities, which appear to be integral multiples of 96 cm/s. The velocities apparently depend on the species, environment, and type of pulse when changed artificially. Measured vertical velocities are larger than horizontal velocities. This may provide a basis for gravitropism and may suggest a cosmic connection. Mean ratios of reciprocals of horizontal and vertical internodal spacings, in simple structure cases, give ratios like 1.25, 1.33, 1.50, 1.66, 3.0, and likely others. These ratios appear to confirm the velocity measurements since ratios of integral multiples of 96 cm/s give the same ratios. The versatility of plant growth is enhanced by the velocity variability.     

Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging

Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin.     

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.     

Kinetin-induced Formation of Gametophores in Dark Cultures of Ceratodon purpureus

The effects of various concentrations of kinetin on the developmentof gametophytes were studied in light and dark cultures of Ceratodonpurpureus in liquid media. There was no bud formation in eitherlight or dark control cultures. Buds were produced under theinfluence of kinetin in light as well as in dark cultures, theeffect of a given concentration of kinetin being a little strongerin the light. Kinetin has been thus found to be an essential factor of budformation in a moss which hardly produces gametophores evenin the light. This is believed to be the first report of gametophore formationin the dark since Keil's short communication in 1949. It alsorepresents a new example replacing a morphogenetic light stimulusby kinetin.