Cell Wall Assembly and Intracellular Trafficking in Plant Cells Are Directly Affected by Changes in the Magnitude of Gravitational Acceleration

Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.     

Calcium balance changes in tip growing plant cells under clinorotation

Calcium is known to play an important role in the regulation of cellular processes as a secondary messenger in signal transduction to a specific response in all eucaryotic cells. Its messenger role is realized by transient changes in the Ca2+ cytosolic concentration induced by variety of external stimuli such as light, hormones and gravity. Recent findings claim the modifications in a calcium balance in plant cells in microgravity and under clinorotation reproducing partially the microgravity effect. Based on these data, the hypothesis is proposed that Ca2+ level changes can trigger the rearrangements in cell metabolism occurring in the conditions of altered gravity. However, the methods used in previous works permit only to determine the relative quantity of intracellular membrane-bound calcium and to observe the localization of free Ca2+. Therefore, it is of essential interest to measure the concentration of free calcium ions ([Ca2+]i) in plant cells under the influence of altered gravity. In this paper results from measurements of the [Ca2+]i in plant cells under clinorotation will be reviewed and discussed in an effort to examine their effectiveness, causes and consequences.     

Asymmetric cytokinin signaling opposes gravitropism in roots

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.     

Altered gravity affects subnucleolus localization of fibrillarin and NopA64, the most important proteins of rRNA processing

Fibrillarin and plant nucleolin homologue NopA64 are two important nucleolar proteins involved in pre-rRNA processing. To understand better the effects of the altered gravity environment on the nucleolus functioning we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under simulated microgravity that was compared to the control cells grown in normal conditions at I g. Cress fibrillarin was first shown to have the molecular weight 41 kDa. Both fibrillarin and NopA64 in the cress cell nucleolus are located in the zones known to contain processing pre-rRNA molecules as it has been previously reported in other species. The data confirm participation of these proteins in processomes--RNP complex particles involved in pre-rRNA processing. Under altered gravity a decrease in the quantity of both fibrillarin and NopA64 in the transition zone between fibrillar centres and the dense fibrillar component was observed, compared to control, which could point out to a lowering of the level of early pre-rRNA processing in these experimental conditions. This decrease was also detected in the bulk of the dense fibrillar component. These data support the idea that altered (reduced) gravity results in lowering the level of functional activity of the nucleolus.     

微重力及模拟微重力对植物生长的影响

重力对地球上生物的生长、发育、代谢及繁殖等具有重要影响.植物细胞的重力敏感性已被众多研究所证明,在空间微重力环境或地面模拟微重力环境下,植物表现特殊的微重力反应.微重力或模拟微重力会对植物体生长产生一系列的影响.综述微重力及模拟微重力对植物生长的影响,并对近期这一领域的研究进行了概括.     

Functional aspects of cell patterning in aerial epidermis

Plants have evolved epidermal cells that have specialized functions as adaptations to life on land. Many of the functions of these specialized cells are dependent, to a significant extent, on their arrangement within the aerial epidermis. Considerable progress has been made over the past two years in understanding the patterning mechanisms of trichomes and stomata in Arabidopsis leaves at the molecular level. How universal are these patterning programmes, and how are they adjusted to meet the changing functions of specialized epidermal cells in different plant organs? In this review, we compare the patterning of stomata and trichomes in different plant species, describe environmental and developmental factors that alter cell patterning, and discuss how changes in patterning might relate to cell function. Patterning is an important aspect to the functioning of aerial epidermal cells, and a greater understanding of the processes that are involved will significantly enhance our understanding of how cellular activities are integrated in multicellular plants.     

Joining forces: The interface of gravitropism and plastid protein import

In flowering plants, gravity perception appears to involve the sedimentation of starch-filled plastids, called amyloplasts, within specialized cells (the statocytes) of shoots (endodermal cells) and roots (columella cells). Unfortunately, how the physical information derived from amyloplast sedimentation is converted into a biochemical signal that promotes organ gravitropic curvature remains largely unknown. Recent results suggest an involvement of the Translocon of the Outer Envelope of (Chloro) plastids (TOC) in early phases of gravity signal transduction within the statocytes. This review summarizes our current knowledge of the molecular mechanisms that govern gravity signal transduction in flowering plants and summarizes models that attempt to explain the contribution of TOC proteins in this important behavioral plant growth response to its mechanical environment.Key words: gravitropism, root, amyloplast, TOC complex, TOC132, TOC75     

The effect of simulated microgravity on osteoblasts is independent of the induction of apoptosis

Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non-rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3-E1 osteoblast-like cells for 1-14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T-1/2 stem cells, MC3T3-E1 osteoblast-like cells, or MLO-A5 osteocyte-like cells to the action of these agents. RT-PCR analysis indicated that MC3T3-E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector-averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death.     

Changes in gravity rapidly alter the magnitude and direction of a cellular calcium current

In single-celled spores of the fern Ceratopteris richardii, gravity directs polarity of development and induces a directional, trans-cellular calcium (Ca²⁺) current. To clarify how gravity polarizes this electrophysiological process, we measured the kinetics of the cellular response to changes in the gravity vector, which we initially estimated using the self-referencing calcium microsensor. In order to generate more precise and detailed data, we developed a silicon microfabricated sensor array which facilitated a lab-on-a-chip approach to simultaneously measure calcium currents from multiple cells in real time. These experiments revealed that the direction of the gravity-dependent polar calcium current is reversed in less than 25 s when the cells are inverted, and that changes in the magnitude of the calcium current parallel rapidly changing g-forces during parabolic flight on the NASA C-9 aircraft. The data also revealed a hysteresis in the response of cells in the transition from 2g to micro-g in comparison to cells in the micro-g to 2-g transition, a result consistent with a role for mechanosensitive ion channels in the gravity response. The calcium current is suppressed by either nifedipine (calcium-channel blocker) or eosin yellow (plasma membrane calcium pump inhibitor). Nifedipine disrupts gravity-directed cell polarity, but not spore germination. These results indicate that gravity perception in single plant cells may be mediated by mechanosensitive calcium channels, an idea consistent with some previously proposed models of plant gravity perception.     

Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity

Summary. Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus. Correspondence and reprints: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis . Previous studies have shown that the arc 6 and arc 12 ( a ccumulation and r eplication of c hloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc 6 and arc 12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc 6 and arc 12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc 6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.     

The interaction of microgravity and ethylene on the ultrastructure cell and Ca2+ localization in soybean hook hypocotyl

Calcium ions are secondary messenger in numerous cellular processes of plant grown at 1 g. Ca2+ are connected with oxygen atoms, of pectin carboxy groups and/or with H(+)-groups of protein (Roux and Slocum, 1982; Hepler and Wayne, 1985). The influence of altered gravity on the calcium balance in some cells is established. The increased synthesis of ethylene in plant grown in microgravity caused the change of the structural-functional organization of cell (Hensel and Iversen, 1980; Hilaire et al., 1996). Available data put the new question: how do high ethylene level and microgravity influence on the redistribution of Ca2+ in cell of seedling in early stage of growth? Therefore, the goal of our data was the comparable study of the cell ulltrastructure and localization of Ca2+ in hook hypocotyl of soybean seedling under interaction of microgravity and ethylene.     

The primary effects of clinorotation on cultured human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are specific cells capable of long-term proliferation and differentiation into various stromal tissue cell types. The state of MSCs depends on the cellular microenvironment and several soluble factors. We proposed that gravity could, in addition, influence MSCs features. To prove this hypothesis, we studied the effects of prolonged clinorotation on cultured human MSC morphology, proliferation rate and expression of specific cellular markers. Human bone marrow-derived MSCs were isolated by Histopaque-1.077 density centrifugation and cultured in DMEM-LG with 10% FBS. MSC cultures were composed of fibroblastoid cells negative for hemopoietic cell markers and positive for ASMA, collagen-1, fibronectin, CD54, CD105 and CD106. Cells were exposed to clinorotation from 1 hour to 10 days. It was shown that the proliferative rate was decreased in experimental cultures as compared to cells growing in normal conditions. Clinorotated MSCs appeared more flattened and reached confluence at a lower cell density. The obtained results suggest that cultured human mesenchymal stem cells sense the changes in gravity vector and may respond to microgravity by altered functional activity.     

Microgravity environment uncouples cell growth and cell proliferation in root meristematic cells: The mediator role of auxin

Experiments performed in space have evidenced that, in root meristematic cells, the absence of gravity results in the uncoupling of cell growth and cell proliferation, two essential cellular functions that support plant growth and development, which are strictly coordinated under normal ground gravity conditions. In space, cell proliferation appears enhanced whereas cell growth is depleted. Since coordination of cell growth and proliferation is a major feature of meristematic cells, the observed uncoupling is a serious stress condition for these cells producing important alterations in the developmental pattern of the plant. Auxin plays a major role in these processes both by assuring the coupling of cell growth and proliferation under normal conditions and by exerting a decisive influence in the uncoupling under altered gravity conditions. Auxin is a mediator of the transduction of the gravitropic signal and its distribution in the root is altered subsequent to a change in the gravity conditions. This altered distribution may produce changes in the expression of specific growth coordinators leading to the alteration of cell cycle and protein synthesis. Therefore, available data indicate that the effects of altered gravity on cell growth and proliferation are the consequence of the transduction of the gravitropic signal perceived by columella cells, in the root tip.Key words: cell cycle, ribosome biogenesis, nucleolus, auxin efflux, graviperception, space flight, arabidopsisThe size and morphology of plants and of plant organs is basically determined by cellular activities that occur in meristems. The primary meristems are root and shoot apical meristems, located at both upper and lower ends of the plant, which are constituted by stem cells. Cell division in these meristems is required to supply new cells for expansion and differentiation of tissues and initiation of new organs, providing the basic structure of the plant body.¹ In turn, active protein synthesis is required after mitosis in order to promote the necessary cell growth, up to duplication of cell size, which will make possible a new cell division. This continuous activity of growth and proliferation in meristematic cells is controlled by auxin, whose distribution in roots sets up distinct zones for cell division, cell expansion and differentiation and determines the balance between them.^2,3Therefore, cell growth and proliferation are essential functions for plant development and they are involved in the developmental response to environmental stimuli, such as tropisms and defense mechanisms against both biotic and abiotic agents.^4–6 Gravity is a fundamental environmental condition, constant in the Earth as a factor conditioning life throughout its whole history. Plants are particularly affected by gravity in their growth, which is directed by the gravity vector producing the well known process of gravitropism.An experiment aimed to know the effects of a weightless environment on cell proliferation and growth in root meristematic cells was performed in the International Space Station. It consisted of germinating seeds of Arabidopsis thaliana in space and then growing seedlings for four days at the constant temperature of 22°C, in the darkness. Seedlings were fixed when still in space and recovered on ground to be processed for microscopical study. In addition, samples from a previous space experiment, grown in a similar way but fixed differently and including a control flight experiment in a 1 g centrifuge, were also incorporated to the analysis.^7,8 This analysis consisted of biometrical estimations of the seedling and root length, quantitative measurements at the cellular level, including number of cells per millimeter in specific cell files, in order to get an estimate of the cell proliferation rate, and morphometrical, ultrastructural and immunocytochemical study of the nucleolus, in order to know the rate of ribosome biogenesis, as an estimation of the level of protein synthesis, which is the cellular process which determines cell growth in the root meristem. Data obtained from space-flown samples were compared with 1 g ground controls and also with data from samples grown in the same conditions in a device called “Random Positioning Machine”, an efficient simulator of microgravity, which induces constant changes of the gravity vector as it is sensed by living samples.⁹ The results interestingly showed an enhanced rate of cell proliferation accompanied by a reduction of ribosome biogenesis per cell in samples grown in both real and simulated microgravity, compared to 1 g controls, either in flight or on ground.¹⁰ This alteration of essential cellular processes may go far beyond the mere change in specific physiological activities of a particular cell type, since, on the one hand, alteration of cell growth and proliferation in the root meristem may have consequences at the level of development and shaping of the whole plant; on the other hand, regulation of these cellular activities by auxin may put in connection these cellular alterations with the transduction cascade of the gravitropic signal perceived by columella cells in the root tip, which is altered when the environmental gravity conditions change and which finally results in the modification of the levels and distribution of auxin throughout the root.     

Signal transduction in cells of the immune system in microgravity

Life on Earth developed in the presence and under the constant influence of gravity. Gravity has been present during the entire evolution, from the first organic molecule to mammals and humans. Modern research revealed clearly that gravity is important, probably indispensable for the function of living systems, from unicellular organisms to men. Thus, gravity research is no more or less a fundamental question about the conditions of life on Earth. Since the first space missions and supported thereafter by a multitude of space and ground-based experiments, it is well known that immune cell function is severely suppressed in microgravity, which renders the cells of the immune system an ideal model organism to investigate the influence of gravity on the cellular and molecular level. Here we review the current knowledge about the question, if and how cellular signal transduction depends on the existence of gravity, with special focus on cells of the immune system. Since immune cell function is fundamental to keep the organism under imnological surveillance during the defence against pathogens, to investigate the effects and possible molecular mechanisms of altered gravity is indispensable for long-term space flights to Earth Moon or Mars. Thus, understanding the impact of gravity on cellular functions on Earth will provide not only important informations about the development of life on Earth, but also for therapeutic and preventive strategies to cope successfully with medical problems during space exploration.     

Differentiation in plant epidermal cells

The plant epidermis is a multifunctional tissue playing important roles in water relations, defence and pollinator attraction. This range of function is performed by a number of different types of specialized cells, which differentiate from the early undifferentiated epidermis in adaptively significant patterns and frequencies. These various cells show different degrees of morphological specialization, but there is evidence to suggest that even the less specialized cell types may require certain signals to ensure their correct differentiation and patterning. Epidermal cells may potentially adopt certain fates through a cell lineage based mechanism or a cell interaction mechanism. Work on stomatal development has focused on the cell lineage mechanism and work on trichome differentiation has focused on the cell interaction model. Recent work on the Arabidopsis trichome suggests that interactions between neighbouring cells reinforce initial differences, possibly in levels of gene expression or cell cycle stage, to commit cells to different developmental programmes. In this review these mechanisms are explored in a number of specialized cell types and the further interactions between different developmental programmes are analysed. It is in these interactions between differentiating cells adopting different cell fates that the key to the patterning of a multifunctional tissue must lie.     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

Effects of microgravity on cell cytoskeleton and embryogenesis

The aim of this review is to compile, summarize and discuss the effects of microgravity on embryos, cell structure and function that have been demonstrated from data obtained during experiments performed in space or in altered gravity induced by clinostats. In cells and tissues cellular structure and genetic expression may be changed in microgravity and this has a variety of effects on embryogenesis which include death of the embryo, failure of neural tube closure, or final deformities to the overall morphology of the newborn or hatchling. Many species and protocols have been used for microgravity space experiments making it difficult to compare results. Experiments on the ways in which embryonic development and cell interactions occur in microgravity could also be performed. Experiments that have been done with cells in microgravity show changes in morphology, cytoskeleton and function. Changes in cytoskeleton have been noted and studies on microtubules in gravity have shown that they are gravity sensitive. Further study of basic chemical reactions that occur in cells should be done to shed some light on the underling processes leading to the changes that are observed in cells and embryos in microgravity.