Conservation of the plastid sedimentation zone in all moss genera with known gravitropic protonemata.

Moss protonemata from several species are known to be gravitropic. The characterization of additional gravitropic species would be valuable to identify conserved traits that may relate to the mechanism of gravitropism. In this study, four new species were found to have gravitropic protonemata, Fissidens adianthoides, Fissidens cristatus, Physcomitrium pyriforme, and Barbula unguiculata. Comparison of upright and inverted apical cells of P. pyriforme and Fissidens species showed clear axial sedimentation. This sedimentation is highly regulated and not solely dependent on amyloplast size. Additionally, the protonemal tip cells of these species contained broad subapical zones that displayed lateral amyloplast sedimentation. The conservation of a zone of lateral sedimentation in a total of nine gravitropic moss species from five different orders supports the idea that this sedimentation serves a specialized and conserved function in gravitropism, probably in gravity sensing.

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.

Gravity sensing in moss protonemata.

Moss protonemata are a valuable system for studying gravitropism because both sensing and upward curvature (oriented tip growth) take place in the same cell. We review existing evidence, especially for Ceratodon purpureus, that addresses whether the mass that functions in sensing is that of amyloplasts that sediment. Recent experiments show that gravitropism can take place in media that are denser than the apical cell. This indicates that gravity sensing relies on an intracellular mass rather than that of the entire cell and provides further support for the starch-statolith hypothesis of sensing. Possible mechanisms for how amyloplast mass functions in sensing and transduction are discussed.

Autonomic straightening of gravitropically curved cress roots in microgravity.

The typical response of plant organs to gravistimulation is differential growth that leads to organ bending. If the gravitropic stimulus is withdrawn, endogenous compensation of the graviresponse and subsequent straightening occur in some plants. For instance, autonomic straightening of Lepidium roots occurs when gravitropically-curved rootsare rotated on a clinostat (Stankovi et al., 1998a). To determine whether endogenous compensation of the graviresponse also occurs in space, microgravity-grown cress roots were laterally centrifuged in-flight and then returned to microgravity using Biorack hardware on a shuttle mission (STS-81). The cress roots were centrifuged at 4 different g-doses (0.1 x g and 1 x g for 15 or 75 min). All four treatments yielded varying degrees of root curvature. Upon removal from the centrifuge, roots in all four treatments underwent subsequent straightening in microgravity. This straightening resulted from a loss of gravitropic curvature in older regions of the root and the coordinated alignment of new growth. These results show that both microgravity and clinostat rotation on Earth are equivalent in stimulus withdrawal with respect to the induction of endogenous compensation of the curvature. Cress roots are the only plant organ shown to undergo compensation of the curvature in both microgravity and on a clinostat. The compensation of graviresponse in space rules out the hypothesis that the endogenous root straightening ("autotropism") represents a commitment to a pre-stimulus orientation with respect to gravity and instead suggests that there is a default tendency towards axiality following a withdrawal of a g-stimulus.

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.

Oriented asymmetric divisions that generate the stomatal spacing pattern in arabidopsis are disrupted by the too many mouths mutation.

Wild-type stomata are spaced by intervening cells, a pattern disrupted in the Arabidopsis mutant too many mouths (tmm). To determine the mechanism of wild-type spacing and how tmm results in pattern violations, we analyzed the behavior of cells through time by using sequential dental resin impressions. Meristemoids are stomatal precursors produced by asymmetric division. We show that wild-type patterning largely results when divisions next to a preexisting stoma or precursor are oriented so that the new meristemoid is placed away. Because this placement is independent of cell lineage, these divisions may be oriented by cell-cell signaling. tmm randomizes this orientation and releases a prohibition on asymmetric division in cells at specific locations, resulting in stomatal clusters. TMM is thus necessary for two position-dependent events in leaves: the orientation of asymmetric divisions that pattern stomata, and the control of which cells will enter the stomatal pathway. In addition, our findings argue against most previous hypotheses of wild-type stomatal patterning.

Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants.

Root gravitropism in wild-type Arabidopsis and in two starchless mutants, pgm1-1 and adg1-1, was evaluated as a function of light position to determine the relative strengths of negative phototropism and of gravitropism and how much phototropism affects gravitropic measurements. Gravitropism was stronger than phototropism in some but not all light positions in wild-type roots grown for an extended period, indicating that the relationship between the two tropisms is more complex than previously reported. Root phototropism significantly influenced the time course of gravitropic curvature and the two measures of sensitivity. Light from above during horizontal exposure overestimated all three parameters for all three genotypes except the wild-type perception time. At the irradiance used (80 micromol m(-2) s(-1)), the shortest periods of illumination found to exaggerate gravitropism were 45 min of continuous illumination and 2-min doses of intermittent illumination. By growing roots in circumlateral light or by gravistimulating in the dark, corrected values were obtained for each gravitropic parameter. Roots of both starchless mutants were determined to be about three times less sensitive than prior estimates. This study demonstrates the importance of accounting for phototropism in the design of root gravitropism experiments in Arabidopsis.

The density of apical cells of dark-grown protonemata of the moss Ceratodon purpureus.

Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the moss Ceratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm3) were considerably lower than from other methods (1.046 to 1.085 g/cm3). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.

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 > or = 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-1 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.

Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves.

Stomatal development was studied in wild-type Arabidopsis leaves using light and electron microscopy. Development involves three successive types of stomatal precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell wall patterns indicates that meristemoids can divide asymmetrically a variable number of times. Before meristemoid division, the nucleus and a preprophase band of microtubules become located on one side of the cell, and the vacuole on the other. Meristemoids are often triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells. Because these features are also found in developing stomata, stomatal differentiation begins in GMCs. The wall thickenings mark the division site in the GMC since they overlie a preprophase band of microtubules and occur where the cell plate fuses with the parent cell wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidney-shaped guard cells. This identification of stages in stomatal development in wild-type Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective in stomatal patterning, cell specification, and differentiation.

Curvature induced by amyloplast magnetophoresis in protonemata of the moss Ceratodon purpureus.

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm-3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal

Red light-induced suppression of gravitropism in moss protonemata.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.

Spaceflight hardware allowing unilateral irradiation and chemical fixation in petri dishes.

To accommodate a spaceflight experiment with moss (SPM), experiment-unique equipment (EUE) was developed by engineers at Kennedy Space Center. The hardware allows sterile culture for an extended period of time in commercial petri dishes, lateral illumination of each culture with light of a specific wavelength (660 nm; other wavelengths are possible) and a range of intensities (0.05-5 micromoles photons m-2 s-1), incubation in complete darkness, and chemical fixation to terminate the experiment under conditions of microgravity. The use of a fixative required triple containment to protect the astronaut crew. An external panel on the experiment container allowed the timing of illumination and fixation to be controlled by the crew. Light quality is provided by light emitting diodes (LEDs) that are located in the lid of the outer container, the BRIC (Biological Research In Canisters)-LED. Each canister accommodates 6 Petri Dish Fixation Units (PDFUs), and each PDFU holds one 6 cm petri dish. All components are autoclavable. LED illumination is piped through a transparent glass rod. Each PDFU contains fixative in a reservoir that is released by the depression of an actuator. This hardware performed well during its first flight, the 16-day STS-87 mission in Nov./Dec., 1997 as part of the Collaborative USA and Ukrainian Experiment (CUE). It supported vigorous and sterile moss growth, cells were maintained in position and were well-fixed, and there was a vigorous and consistent response to light. Although here used for moss, in future flight experiments this unique new hardware can be used for many types of organisms normally grown in petri dishes, with or without a requirement for illumination.

Divergent regulation of stomatal initiation and patterning in organ and suborgan regions of the Arabidopsis mutants too many mouths and four lips.

Stomata are consistently patterned so that they are not in contact. This patterning is violated in the too many mouths (tmm) and four lips (flp) mutations of Arabidopsis thaliana (L.) Heynh. which have stomatal clusters in the first-formed leaves. To clarify the function of both genes in stomatal initiation and patterning, the phenotypes of many different organs were quantified. The flp mutation affects dorsiventral and cylindrical organs differentially with respect to the frequency of clustering. The tmm mutation has a more complex region-specific phenotype in that some regions lack stomata entirely, other regions have excess stomata, and the flower stalk exhibits an apex-to-base gradient from excess to no stomata. This suggests that TMM represents an unusual type of gene regulating plant cell development in that it can either influence stomatal initiation in a positive or negative fashion depending on region. Since the frequencies of initiation and clustering can be uncoupled in tmm, these two functions are under separate region-specific control. Analysis of double mutants shows that tmm and flp in some cases show region-specific interactions in both cluster formation and initiation, and that there may be subpopulations of stomata under different genetic control.

Autonomic straightening after gravitropic curvature of cress roots.

Few studies have documented the response of gravitropically curved organs to a withdrawal of a constant gravitational stimulus. The effects of stimulus withdrawal on gravitropic curvature were studied by following individual roots of cress (Lepidium sativum L.) through reorientation and clinostat rotation. Roots turned to the horizontal curved down 62 degrees and 88 degrees after 1 and 5 h, respectively. Subsequent rotation on a clinostat for 6 h resulted in root straightening through a loss of gravitropic curvature in older regions and through new growth becoming aligned closer to the prestimulus vertical. However, these roots did not return completely to the prestimulus vertical, indicating the retention of some gravitropic response. Clinostat rotation shifted the mean root angle -36 degrees closer to the prestimulus vertical, regardless of the duration of prior horizontal stimulation. Control roots (no horizontal stimulation) were slanted at various angles after clinostat rotation. These findings indicate that gravitropic curvature is not necessarily permanent, and that the root retains some commitment to its equilibrium orientation prior to gravitropic stimulation.

Gravitropism and gravimorphism during regeneration from protoplasts of the moss Ceratodon purpureus (Hedw.) Brid.

Wild-type (WT) protonemata of the moss Ceratodon purpureus grow upwards in darkness (negative gravitropism), whereas protonemata of the mutant, wrong-way response (wwr-1) grow down. Since Ceratodon protoplasts regenerate to form new protonemata, we analyzed whether the direction of filament emergence was influenced by gravity (gravimorphism) and determined the cytological events that correlated with the onset of gravitropism in WT and wwr-1 filaments formed de novo. In the WT the direction of filament emergence appeared to be gravimorphic as more than 66% of the new filaments emerged above the horizontal. In contrast, the direction of filament emergence was random in wwr-1. Tip-growing cells of both genotypes became gravitropic within a total of one to two cell divisions. Gravitropic curvature in wwr-1 was opposite in direction to that of WT, and the timing of curvature was comparable, indicating that the wwr-1 mutation acts during the onset of gravitropic competence. In time-lapse studies of both genotypes, neither a plastid-free zone nor obvious and extensive plastid sedimentation characteristic of mature dark-grown protonemata was observed in the new filaments prior to gravitropic curvature. Thus, it appears that these latter two features are not required for gravitropism in new protonemal filaments from protoplasts.

Light promotion of hypocotyl gravitropism of a starch-deficient tobacco mutant correlates with plastid enlargement and sedimentation.

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.

Gravitropism in caulonemata of the moss Pottia intermedia.

The gravitropism of caulonemata of Pottia intermedia is described and compared with that of other mosses. Spore germination produces primary protonemata including caulonemata which give rise to buds that form the leafy moss plant, the gametophore. Primary caulonemata are negatively gravitropic but their growth and the number of filaments are limited in the dark. Axenic culture of gametophores results in the production of secondary caulonemata that usually arise near the leaf base. Secondary protonemata that form in the light are agravitropic. Secondary caulonemata that form when gametophores are placed in the dark for several days show strong negative gravitropism and grow well in the dark. When upright caulonemata are reorientated to the horizontal or are inverted, upward bending can be detected after 1 h and caulonemata reach the vertical within 1-2 d. Clear amyloplast sedimentation occurs 10-15 minutes after horizontal placement and before the start of upward curvature. This sedimentation takes place in a sub-apical zone. Amyloplast sedimentation also takes place along the length of upright and inverted Pottia protonemata. These results support the hypothesis that amyloplast sedimentation functions in gravitropic sensing since sedimentation occurs before gravitropism in Pottia and since the location and presence of a unique sedimentation zone is conserved in all four mosses known to gravitropic protonomata.

Growth of Pottia intermedia protonemata in altered gravity.

Plants are immobile; therefore, they are oriented in space due to growth movements--tropisms. The latter occur in response to environmental stimuli such as gravity (gravitropism), light (phototropism), chemical compounds or water (chemo- and hydrotropisms). Gravity is the only force that was impossible to control. The moss protonemata are among the limited group of plant objects with tip growth. What is unique about this structure is that protonemal apical cells both sense and respond to gravity. It is considered that the apical cell perceives gravity through amyloplasts (Sack, 1993; Chaban, 1996). Although the dynamics of protonemata negative gravitropism in different moss species was studied in detail, the role of gravity in both the structural polarity of apical cells and the formation of protonematal mat with circular symmetry is completely unexplored. Using the unique possibility to fly the moss on the space shuttle (STS-87) we aimed in this study to analyze the character of the interaction of gravity with light and endogenous factors in the pattern of protonemata space orientation.