Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.     

An active role of the amyloplasts and nuclei of root statocytes in graviperception.

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception.     

Effects of slow clinorotation on lipid contents and proton permeability of thylakoid membranes of pea chloroplasts.

Photochemical characteristics and lipid composition of thylakoid membranes from 12 day-old pea leaves that were exposed to slow clino-rotation were examined and compared with a vertical control. Proton permeability of thylakoid membranes was estimated from light-induced proton uptake (delta H+) and post-illumination proton efflux in chloroplast suspensions. The delta pH magnitude was calculated from the level of light-induced quenching of 9-aminoacridine fluorescence. Proton permeability of thylakoid membranes increased during exposure to clino-rotation. When subsequently transferred to darkness, proton efflux increased almost 2-fold in clinorotated leaves. The results were compared with data on pigment and polar lipid composition of photosynthetic membranes in clino-rotated and control plants. It was concluded that both the increase of proton permeability and the decrease of polar lipid content in chloroplasts were induced by clino-rotation.     

Patterns of cortical microtubules formed in epidermis of Beta vulgaris L. roots under clinorotation.

Changes of cortical microtubules (MTs) from the normal transverse arrangement were observed in epidermal cells of Beta vulgaris roots under clinorotation. We hypothesize that the epidermis is sensitive to clinorotation and that the microtubular cytoskeleton plays a key role in the ensuing growth response.     

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.     

植物细胞响应重力变化的Ca2+信号

重力大小的改变(微重力和超重力)可以对植物的生长发育、生理生化特性、细胞超微结构、基因和蛋白的表达等产生广泛的影响,而Ca~(2+)可能在此过程中起信号物质的作用.重力刺激在植物细胞中引发事件的顺序可能是:重力刺激的感受—细胞膜系统张力改变—膜理化特性改变—膜透性、离子转运、膜连接酶活性等改变—Ca~(2+)信号的产生和转导—新陈代谢变化—生理反应.植物对重力水平变化应激响应的至关重要一步是引起细胞内Ca~(2+)分布区域和浓度的变化,这是将细胞外重力刺激转换为细胞内化学信号的关键步骤.由于机械力敏感的C~(2+)通道的活化和Ca~(2+)-ATPase酶活性受到抑制,重力改变时细胞质中自由Ca~(2+)浓度增加,随后Ca~(2+)作为第二信使介导相关酶活性发生改变,最终引起一连串的生理生化反应.本文探讨了重力变化对植物细胞质内自由Ca~(2+)浓度的影响、Ca~(2+)信号的产生机制.以及Ca~(2+)作为次级信号对细胞生理生化过程调节作用的途径和机制,介绍了常用的Ca~(2+)研究方法,并分析了研究的关键点和难点.     

Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in urodeles.

Two species of newts (Urodela) and two types of clinostats for fast clinorotation (60 rpm) were used to investigate the influence of simulated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, differentiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with Triturus vulgaris using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles waltl using both horizontal and radial clinostats the results were contradictory compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictory results.     

Functional state of chromatin and proliferative activity of meristematic cells in pea seedlings under various clinostatic conditions.

Data from a complex cytochemical analysis show that the functional state of chromatin and the level of the cell proliferative activity may be reliable cytological criteria for primary structural and functional changes that result in disturbances of plant growth and development. Autoradiographic and cytophotonetric studies made it possible to establish certain differences, induced by fast rotation (50 rev/min) on the clinostat, in the chromatin state and cell reproduction of the pea seedling root meristem for the initial stages of plant development. There were no essential differences for the given parameters under slow (2 rev/min) clinostatic conditions.     

Temporal regulation of global gene expression and cellular morphology in Xenopus kidney cells in response to clinorotation.

Here, we report changes gene expression and morphology of the renal epithelial cell line, A6, which was derived from Xenopus laevis adult kidney that had been induced by long-term culturing with a three-dimensional clinostat. An oligo microarray analysis on the A6 cells showed that mRNA levels for 52 out of 8091 genes were significantly altered in response to clinorotation. On day 5, there was no dramatic change in expression level, but by day 8 and day 10, either upregulation or downregulation of gene expression became evident. By day 15, the expression levels of 18 out of 52 genes had returned to the original levels, while the remaining 34 genes maintained the altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in the mRNA levels of selected genes were found only under clinorotation and not under hypergravity (7 g) or ground control. Morphological changes including loss of dome-like structures and disorganization of both E-cadherin adherence junctions and cortical actin were also observed after 10 days of culturing with clinorotation. These results revealed that the expression of selected genes was altered specifically in A6 cells cultured under clinorotation.     

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.     

Plant growth and gas balance in a plant and mushroom cultivation system.

In order to obtain basic data for construction of a plant cultivation system incorporating a mushroom cultivation subsystem in the CELSS, plant growth and atmospheric CO2 balance in the system were investigated. The plant growth was promoted by a high level of CO2 which resulted from the respiration of the mushroom mycelium in the system. The atmospheric CO2 concentration inside the system changed significantly due to the slight change in the net photosynthetic rate of plants and/or the respiration rate of the mushroom when the plant cultivation system combined directly with the mushroom cultivation subsystem.     

Material balance and diet in bioregenerative life support systems: connection with coefficient of closure.

Bioregenerative life support systems (BLSS) with different coefficients of closure are considered. The 66.2% coefficient of closure achieved in "BIOS-3" facility experiments has been taken as a base value. The increase in coefficient of closure up to 72.6-93.0% is planned due to use of soil-like substrate (SLS) and concentrating of urine. Food values were estimated both in a base variant ("BIOS-3"), and with increases in the coefficient of closure. It is shown that food requirements will be more fully satisfied by internal crop production with an increase in the coefficient of closure of the BLSS. Changes of massflow rates on an 'input-output' and inside BLSS are considered. Equations of synthesis and degradation of organic substances in BLSS were examined using a stoichiometric model. The paper shows that at incomplete closure of BLSS containing SLS there is a problem of nitrogen balancing. To compensate for the removal of nitrogen from the system in urine and feces, it is necessary to introduce food and a nitrogen-containing additive.     

Effects of air velocity on photosynthesis of plant canopies under elevated CO2 levels in a plant culture system.

To obtain basic data for adequate air circulation for promoting plant growth in closed plant production modules in bioregenerative life support systems in space, effects of air velocities ranging from 0.1 to 0.8 m s-1 on photosynthesis in tomato seedlings canopies were investigated under atmospheric CO2 concentrations of 0.4 and 0.8 mmol mol-1. The canopy of tomato seedlings on a plug tray (0.4 x 0.4 m2) was set in a wind-tunnel-type chamber (0.6 x 0.4 x 0.3 m3) installed in a semi-closed-type assimilation chamber (0.9 x 0.5 x 0.4 m3). The net photosynthetic rate in the plant canopy was determined with the differences in CO2 concentrations between the inlet and outlet of the assimilation chamber multiplied by the volumetric air exchange rate of the chamber. Photosynthetic photon flux (PPF) on the plant canopy was kept at 0.25 mmol m-2 s-1, air temperature at 23 degrees C and relative humidity at 55%. The leaf area indices (LAIs) of the plant canopies were 0.6-2.5 and plant heights were 0.05-0.2 m. The net photosynthetic rate of the plant canopy increased with increasing air velocities inside plant canopies and saturated at 0.2 m s-1. The net photosynthetic rate at the air velocity of 0.4 m s-1 was 1.3 times that at 0.1 m s-1 under CO2 concentrations of 0.4 and 0.8 mmol mol-1. The net photosynthetic rate under CO2 concentrations of 0.8 mmol mol-1 was 1.2 times that under 0.4 mmol mol-1 at the air velocity ranging from 0.1 to 0.8 m s-1. The results confirmed the importance of controlling air movement for enhancing the canopy photosynthesis under an elevated CO2 level as well as under a normal CO2 level in the closed plant production modules.     

Carbon balance in bioregenerative life support systems: some effects of system closure, waste management, and crop harvest index.

In Advanced Life Support (ALS) systems with bioregenerative components, plant photosynthesis would be used to produce O2 and food, while removing CO2. Much of the plant biomass would be inedible and hence must be considered in waste management. This waste could be oxidized (e.g., incinerated or aerobically digested) to resupply CO2 to the plants, but this would not be needed unless the system were highly closed with regard to food. For example, in a partially closed system where some of the food is grown and some is imported, CO2 from oxidized waste when combined with crew and microbial respiration could exceed the CO2 removal capability of the plants. Moreover, it would consume some O2 produced from photosynthesis that could have been used by the crew. For partially closed systems it would be more appropriate to store or find other uses for the inedible biomass and excess carbon, such as generating soils or growing woody plants (e.g., dwarf fruit trees). Regardless of system closure, high harvest crops (i.e., crops with a high edible to total biomass ratio) would increase food production per unit area and O2 yields for systems where waste biomass is oxidized to recycle CO2. Such interlinking effects between the plants and waste treatment strategies point out the importance of oxidizing only that amount of waste needed to optimize system performance.     

Effect of CO2 and O2 on development and fructification of wheat in closed systems.

The cultivation of wheat (Triticum aestivum L.) was performed in controlled environment chambers with the continuous monitoring of photosynthesis, dark respiration, transpiration and main nutrient uptakes. A protocol in twin chambers was developed to compare the specific effects of low O2 and high CO2. Each parameter is able to influence photosynthesis but different effects are obtained In the development, fructification and seed production, because of the different effects of each parameter on the ratio of reductive to oxidative cycle of carbon. The first main conclusion is that low level of O2, at the same rate of biomass production, strongly acts on the rate of ear appearance and on seed production. Ear appearance was delayed and seed production reduced with a low O2 treatment (approximately 4%). The O2 effect was not mainly due to the repression of the oxidative cycle. The high CO2 treatment (700 to 900 microl l-1) delayed ear appearance by 4 days but did not reduce seed production. High CO2 treatment also reduced transpiration by 20%. Two hypothesis were proposed to explain the similarities and the difference in the O2 and CO2 effects on the growth of wheat.     

CO2 crop growth enhancement and toxicity in wheat and rice.

The effects of elevated CO2 on plant growth are reviewed and the implications for crop yields in regenerative systems are discussed. There is considerable theoretical and experimental evidence indicating that the beneficial effects of CO2 are saturated at about 0.12% CO2 in air. However, CO2 can easily rise above 1% of the total gas in a closed system, and we have thus studied continuous exposure to CO2 levels as high as 2%. Elevating CO2 from 340 to 1200 micromoles mol-1 can increase the seed yield of wheat and rice by 30 to 40%; unfortunately, further CO2 elevation to 2500 micromoles mol-1 (0.25%) has consistently reduced yield by 25% compared to plants grown at 1200 micromoles mol-1; fortunately, there was only an additional 10% decrease in yield as the CO2 level was further elevated to 2% (20,000 micromoles mol-1). Yield increases in both rice and wheat were primarily the result of increased number of heads per m2, with minor effects on seed number per head and seed size. Yield increases were greatest in the highest photosynthetic photon flux. We used photosynthetic gas exchange to analyze CO2 effects on radiation interception, canopy quantum yield, and canopy carbon use efficiency. We were surprised to find that radiation interception during early growth was not improved by elevated CO2. As expected, CO2 increased quantum yield, but there was also a small increase in carbon use efficiency. Super-optimal CO2 levels did not reduce vegetative growth, but decreased seed set and thus yield. The reduced seed set is not visually apparent until final yield is measured. The physiological mechanism underlying CO2 toxicity is not yet known, but elevated CO2 levels (0.1 to 1% CO2) increase ethylene synthesis in some plants and ethylene is a potent inhibitor of seed set in wheat.     

Ultrastructural and growth indices of Chlorella culture in multicomponent aquatic systems under space flight conditions.

Submicroscopic organization of Chlorella cells cultivated under space flight conditions in three-component aquatic system has been studied. Comparison of the experimental cells with that of the controls revealed certain rearrangements of cell organelles particularly, a reduction in the amount of reserve polysaccharides in chloroplasts, increase of cell vacuolization and mitochondrion volume, complication configuration of plasmalemma evaginations and invaginations, and also disturbances in the process of cytokinesis. More over an increase in the number of Chlorella cells infected by bacteria was shown in the experimental variant. No considerable differences were established in the growth characteristics of the experimental and control populations. A comparative cytological analysis revealed general regularities of organelles in Chlorella cells cultivated under space flight condition in the uni- and multicomponent systems.     

Growth and development in higher plants under simulated microgravity conditions on a 3-dimensional clinostat.

Growth and development of etiolated pea (Pisum sativum L. cv. Alaska) and maize (Zea mays L. cv. Golden Cross Bantam) seedlings grown under simulated microgravity conditions were intensively studied using a 3-dimensional clinostat as a simulator of weightlessness. Epicotyls of etiolated pea seedlings grown on the clinostat were the most oriented toward the direction far from cotyledons. Mesocotyls of etiolated maize seedlings grew at random and coleoptiles curved slightly during clinostat rotation. Clinostat rotation promoted the emergence of the 3rd internodes in etiolated pea seedlings, while it significantly inhibited the growth of the 1st internodes. In maize seedlings, the growth of coleoptiles was little affected by clinostat rotation, but that of mesocotyls was suppressed, and therefore, the emergence of the leaf out of coleoptile was promoted. Clinostat rotation reduced the osmotic concentration in the 1st internodes of pea seedlings, although it has little effect on the 2nd and the 3rd internodes. Clinostat rotation also reduced the osmotic concentrations in both coleoptiles and mesocotyls of maize seedlings. Cell-wall extensibilities of the 1st and the 3rd internodes of pea seedlings grown on the clinostat were significantly lower and higher as compared with those on 1 g conditions, respectively. Cell-wall extensibility of mesocotyls in seedlings grown on the clinostat also decreased. Changes in cell wall properties seem to be well correlated to the growth of each organ in pea and maize seedlings. These results suggest that the growth and development of plants is controlled under gravity on earth, and that the growth responses of higher plants to microgravity conditions are regulated by both cell-wall mechanical properties and osmotic properties of stem cells.     

ECOSIMP2 model: prediction of CO2 concentration changes and carbon status in closed ecosystems.

The ECOSIMP2 model, simulating the Plant-Soil-Atmosphere interactions, was developed as a tool for the management of an experimental artificial ecosystem. It consists in three main carbon compartments for production, consumption and decomposition of the biomass. The main biological parameters concern photosynthesis (apparent Km, CO2 compensation point), the harvest index, the rate of consumption, and the kinetics of litter decomposition. From realistic assumptions of kinetics of soil compartments, a steady-state case was obtained, simulating a terrestrial ecosystem. The stability of the atmospheric CO2 concentration was studied after a virtual enclosure of the system in a 20-m high greenhouse. In natural lighting the conditions of stability are severe because of the small size of the atmospheric compartment which amplifies any imbalance between carbon fluxes. The positive consequence of that amplification for research on artificial ecosystems was emphasized.