Automorphosis of higher plants on a 3-D clinostat.

On a three-dimensional (3-D) clinostat, various plant organs developed statocytes capable of responding to the gravity vector. The graviresponse of primary roots of garden cress and maize grown on the clinostat was the same as the control roots, whereas that of maize coleoptiles was reduced. When maize seedlings were grown in the presence of 10(-4) M gibberellic acid and kinetin, the graviresponse of both roots and shoots was suppressed. The corresponding suppression of amyloplast development was observed in the clinostatted and the hormone-treated seedlings. Maize roots and shoots showed spontaneous curvatures in different portions on the 3-D clinostat. The hormone treatment did not significantly influence such an automorphic curvature. When the root cap was removed, maize roots did not curve gravitropically. However, the removal suppressed the automorphic curvatures only slightly. On the other hand, the removal of coleoptile tip did not influence its graviresponse, whereas the spontaneous curvature of decapitated coleoptiles on the clinostat was strongly suppressed. Also, cytochalasin B differently affected the gravitropic and the automorphic curvatures of maize roots and shoots. From these results it is concluded that the graviperception and the early processes of signal transmission are unnecessary for automorphoses under simulated microgravity conditions. Moreover, the results support the view that the amyloplasts act as statoliths probably via an interaction with microfilaments.     

Aquatic food production modules in bioregenerative life support systems based on higher plants.

Most bioregenerative life support systems (BLSS) are based on gravitropic higher plants which exhibit growth and seed generation disturbances in microgravity. Even when used for a lunar or martian base the reduced gravity may induce a decreased productivity in comparison to Earth. Therefore, the implementation of aquatic biomass production modules in higher plant and/or hybrid BLSS may compensate for this and offer, in addition, the possibility to produce animal protein for human nutrition. It was shown on the SLS-89 and SLS-90 space shuttle missions with the C.E.B.A.S.-MINI MODULE that the edible non gravitropic rootless higher aquatic plant Ceratophyllum demeresum exhibits an undisturbed high biomass production rate in space and that the teleost fish species, Xiphophorus helleri, adapts rapidly to space conditions without loss of its normal reproductive functions. Based on these findings a series of ground-based aquatic food production systems were developed which are disposed for utilization in space. These are plant production bioreactors for the species mentioned above and another suitable candidate, the lemnacean (duckweed) species, Wolffia arrhiza. Moreover, combined intensive aquaculture systems with a closed food loop between herbivorous fishes and aquatic and land plants are being developed which may be suitable for integration into a BLSS of higher complexity. Grant numbers: WS50WB9319-3, IVA1216-00588.     

Effect of clinostating on photosynthetic apparatus of pea plants.

The photosynthetic membrane composition and low temperature fluorescence spectra were analyzed for pea chloroplasts from control and clinostated plants. Clinorotation induces a decrease in the amount of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCII) and an increase of its monomeric form. Some changes in organization of photosystem 1 (PS1) complex were revealed as well. These changes are in accordance with the variations of fluorescence characteristics and photochemical activity.     

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.     

Effect of wind velocity on ethylene release from lettuce plants.

Effect of wind velocity on ethylene release rate of intact lettuce plant was investigated. Lettuce plants were grown at wind velocities of 0.1, 0.4, 0.8, and 1.4 m s-1 for 25 to 33 days and then used for ethylene measurement. When ethylene release rate of the plants grown at a wind velocity of 0.1m s-1 was measured at wind velocities of 0.2, 0.6 and 1.0m s-1 the rate was not affected by wind velocity. This result indicates that ethylene diffusion from lettuce leaf to atmosphere is not affected by boundary layer conditions. When ethylene release rate of the plants grown at wind velocities of 0.1, 0.4, 0.8 and 1.4 m s-1 was measured at the same wind velocity as growing conditions, the rate was scarcely increased by high velocity of wind. A strong wind (4.0 m s-1), which induced wounding damage in small areas of the leaves, had no measurable effect on a ethylene release of the whole plant.     

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.     

Use of human wastes oxidized to different degrees in cultivation of higher plants on the soil-like substrate intended for closed ecosystems

To close mass exchange loops in bioregenerative life support systems more efficiently, researchers of the Institute of Biophysics SB RAS (Krasnoyarsk, Russia) have developed a procedure of wet combustion of human wastes and inedible parts of plants using H₂O₂ in alternating electromagnetic field. Human wastes pretreated in this way can be used as nutrient solutions to grow plants in the phototrophic unit of the LSS. The purpose of this study was to explore the possibilities of using human wastes oxidized to different degrees to grow plants cultivated on the soil-like substrate (SLS). The treated human wastes were analyzed to test their sterility. Then we investigated the effects produced by human wastes oxidized to different degrees on growth and development of wheat plants and on the composition of microflora in the SLS. The irrigation solution contained water, substances extracted from the substrate, and certain amounts of the mineralized human wastes. The experiments showed that the human wastes oxidized using reduced amounts of 30% H₂O₂: 1 ml/g of feces and 0.25 ml/ml of urine were still sterile. The experiments with wheat plants grown on the SLS and irrigated by the solution containing treated human wastes in the amount simulating 1/6 of the daily diet of a human showed that the degree of oxidation of human wastes did not significantly affect plant productivity. On the other hand, the composition of the microbiota of irrigation solutions was affected by the oxidation level of the added metabolites. In the solutions supplemented with partially oxidized metabolites yeast-like microscopic fungi were 20 times more abundant than in the solutions containing fully oxidized metabolites. Moreover, in the solutions containing incompletely oxidized human wastes the amounts of phytopathogenic bacteria and denitrifying microorganisms were larger. Thus, insufficiently oxidized sterile human wastes added to the irrigation solutions significantly affect the composition of the microbiological component of these solutions, which can ultimately unbalance the system as a whole.     

Perception of gravity by plants.

Physical principles can be used to predict some features about the gravity perception system in plants. The nature of the system has made it rather elusive, so this approach represents an additional source of information to help find it. For a gravitational stimulus to be detected, two masses must move relative to each other in a manner which causes a significant amount of work to be done on a receptor. Relative to cellular dimensions, the masses must be large, be dense and move noticeable distances. The main sources of noise are thermal motion and flexing of the plant tissue. Some new models for the function of amyloplasts as statoliths are presented.     

Biological effects due to weak magnetic field on plants.

Throughout the evolution process, Earth's magnetic field (MF, about 50 microT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1-1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G1 phase in many plant species (and of G2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stable. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.     

Gravisensing in plants and fungi.

The principle of establishing and maintaining a gravitropic set point angle depends on gravisensing and a subsequent cascade of events that result in differential elongation of the responsive structures. Since gravity acts upon masses, the gravisensing mechanisms of all biological systems must follow the same principle, namely the sensing of some force due to differential acceleration of the perceiving entity and a reference structure. This presentation will demonstrate that gravisensing can be accomplished by various means, ranging from cytoskeletal organization, mechano-elastic stress to perturbation of electric signals. However, several arguments indicate that sedimentation of either dense plastids (statoliths), the entire protoplast, or a combination of these represents the primary step in graviperception in plants. In fungi, nuclei and cytoskeletal proteins are believed to form a network capable of gravisensing but sedimenting organelles that may function as statoliths have been identified. Theoretical and practical limitations of gravisensing and detection of acceleration forces necessitate microgravity experiments to identify the primary perceptor, subsequent biochemical mechano-transduction, and biological response processes.     

Radiosensibility of higher plant seeds after space flight.

The influence of long-term storage of higher plant seeds under space flight conditions (49 to 827 days) on their radiosensibility was studied in the experiments on the orbital stations Salyut 6 and 7. Short-term storage has been proved to have no effect on radiosensitivity of Crepis capillaris seeds. Only in the case of maximal exposure duration the frequency of chromosome aberrations in post-flight irradiated seeds significantly exceeded the chromosome aberration frequency in the ground-based irradiated control. A statistically significant increase in the number of cells with multiple chromosome aberrations was also observed in this experiment. After gamma-irradiation of Arabidopsis thaliana seeds the germinating ability and survival rate of plants decreased depending on the duration of seed storage. Flight-exposed seeds were more sensitive to irradiations with respect to these parameters. A statistically significant increase in the frequency of recessive lethal mutations was observed only in two experiments of long exposure duration.     

The influence of space flight factors on viability and mutability of plants.

The experiments with air-dried Crepis capillaris seeds aboard the Soyuz 16 spaceship and the orbital stations Salyut 5, 6, 7 have revealed an increase in the frequency of aberrant cells in seedlings grown from flight-exposed seeds during the flight (experiment) and after the flight on Earth (flight control) as compared to the ground-based control. The increase in seedlings grown during the flight is more significant than in the flight control. During the flight Arabidopsis thaliana developed from cotyledons to the flowering stage. Analysis of seeds setting on these plants after the flight has shown a reduction in the fertility of these plants and an increase in the frequency of recessive mutants ("Light block-1"). An increased frequency of mutants was also retained in the progeny of plants which had passed through a complete cycle of development during the flight ("Fiton-3"). Suppression of embryo viability was observed in all experiments and expressed itself in reduced germinating ability of seeds from the exposed plants and in the early death of seedlings. Damages resulting from chromosome aberrations are eliminated in the first postflight generation and damages resulting from gene mutations and micro-aberrations are preserved for a longer time.     

Gravitropism of axial organs in multicellular plants.

Gravitropism of plant organs such as roots, stems and coleoptiles can be separated into four distinct phases: 1. perception (gravity sensing), 2. transduction of a signal into the target region and 3. the response (differential growth). This last reaction is followed by a straightening of the curved organ (4.). The perception of the gravitropic stimulus upon horizontal positioning of the organ (1.) occurs via amyloplasts that sediment within the statocytes. This conclusion is supported by our finding that submerged rice coleoptiles that lack sedimentable amyloplasts show no graviresponse. The mode of signal transduction (2.) from the statocytes to the peripheral cell layers is still unknown. Differential growth (3.) consists of a cessation of cell expansion on the upper side and an enhancement of elongation on the lower side of the organ. Based on the facts that the sturdy outer epidermal wall (OEW) constitutes the growth-controlling structure of the coleoptile and that growth-related osmiophilic particles accumulate on the upper OEW, it is concluded that the differential incorporation of wall material (presumably glycoproteins) is causally involved. During gravitropic bending, electron-dense particles ('wall-loosening capacity') accumulate on the growth-inhibited upper OEW. It is proposed that the autotropic straightening response, which is in part due to an acceleration of cell elongation on the curved upper side, may be attributable to an incorporation of the accumulated particles ('release of wall-loosening capacity'). This novel mechanism of autotropic re-bending and its implications for the Cholodny-Went hypothesis are discussed.     

Ontogeny of plants under various gravity condition.

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.     

Evolution of UV-B regulation and protection in plants.

Plants have evolved under the influence of UV-B radiation and have acquired systems for monitoring it and investing appropriate resources for protection against it, i.e., filters, quenchers of radicals and reactive oxygen species, and repair systems. An hypothesis for how plants monitor radiation has been presented.     

Injuries to plants from controlled environment contaminants.

The use of controlled environments is subject to problems from contaminants emitted from materials of the system and from plants. Many contaminants are difficult to identify because injurious dosages are very low, there is a lack of information on what compounds injure plants, because species and cultivars differ greatly in their sensitivity to injury and injury symptoms often are not distinctive. Plastics have been shown to emit many different volatile compounds. The compound, di-butyl phthalate, contained in certain flexible plastics, has been shown to be very toxic to plants. Other injuries have been produced by caulking compounds and bonded screening. Paints have been shown to release xylene that is toxic to plants. Steam for humidification can cause problems because of hydroxylamines and other compounds added to steam used for heating to control fungal growth in return lines. Mercury, from broken thermometers is a particular problem in growth chambers because small quantities can collect in cracks and slowly volatilize to slow growth of plants. Plants themselves release large quantities of volatile hydrocarbons, with ethylene being the commonly recognized chemical that can be damaging when allowed to accumulate. People release large quantities of carbon dioxide which can cause variations in the rate of growth of plants. Contaminant problems can be controlled through filtering of the air or ventilation with make-up air, however the potential for problems is always present and careful testing should be undertaken with the particular species and cultivars being grown insure that there are no toxic agents altering growth in each particular controlled environment being utilized.     

Carbon dioxide exchange of lettuce plants under hypobaric conditions.

Growth of plants in a Controlled Ecological Life Support System (CELSS) may involve the use of hypobaric pressures enabling lower mass requirements for atmospheres and possible enhancement of crop productivity. A controlled environment plant growth chamber with hypobaric capability designed and built at Ames Research Center was used to determine if reduced pressures influence the rates of photosynthesis (Ps) and dark respiration (DR) of hydroponically grown lettuce plants. The chamber, referred to as a plant volatiles chamber (PVC), has a growing area of about 0.2 m2, a total gas volume of about 0.7 m3, and a leak rate at 50 kPa of <0.1%/day. When the pressure in the chamber was reduced from ambient to 51 kPa, the rate of net Ps increased by 25% and the rate of DR decreased by 40%. The rate of Ps increased linearly with decreasing pressure. There was a greater effect of reduced pressure at 41 Pa CO2 than at 81 Pa CO2. This is consistent with reports showing greater inhibition of photorespiration (Pr) in reduced O2 at low CO2 concentrations. When the partial pressure of O2 was held constant but the total pressure was varied between 51 and 101 kPa, the rate of CO2 uptake was nearly constant, suggesting that low pressure enhancement of Ps may be mainly attributable to lowered partial pressure of O2 and the accompanying reduction in Pr. The effects of lowered partial pressure of O2 on Ps and DR could result in substantial increases in the rates of biomass production, enabling rapid throughput of crops or allowing flexibility in the use of mass and energy resources for a CELSS.     

Composition and physical properties of starch in microgravity-grown plants.

The effect of spaceflight on starch development in soybean (Glycine max L., BRIC-03) and potato (Solanum tuberosum, Astroculture-05) was compared with ground controls by biophysical and biochemical measurements. Starch grains from plants from both flights were on average 20-50% smaller in diameter than ground controls. The ratio delta X/delta rho (delta X --difference of magnetic susceptibilities, delta rho--difference of densities between starch and water) of starch grains was ca. 15% and 4% higher for space-grown soybean cotyledons and potato tubers, respectively, than in corresponding ground controls. Since the densities of particles were similar for all samples (1.36 to 1.38 g/cm3), the observed difference in delta X/delta rho was due to different magnetic susceptibilities and indicates modified composition of starch grains. In starch preparations from soybean cotyledons (BRIC-03) subjected to controlled enzymatic degradation with alpha-amylase for 24 hours, 77 +/- 6% of the starch from the flight cotyledons was degraded compared to 58 +/- 12% in ground controls. The amylose content in starch was also higher in space-grown tissues. The good correlation between the amylose content and delta X/delta rho suggests, that the magnetic susceptibility of starch grains is related to their amylose content. Since the seedlings from the BRIC-03 experiment showed elevated post-flight ethylene levels, material from another flight experiment (GENEX) which had normal levels of ethylene was examined and showed no difference to ground controls in size distribution, density, delta X/delta rho and amylose content. Therefore the role of ethylene appears to be more important for changes in starch metabolism than microgravity.     

Lipid peroxidation of plants under microgravity and its simulation.

In series of space experiments aboard the biosatellites "Cosmos 1887", "Bion 9", the orbital stations "Salut", "Mir" and under clinostating, changes of lipid peroxidation (LPO) and antioxidation activity (AOA) of Chlorella, Haplopappus tissue culture, wheat and pea roots were determined. The changes had a complex fluctuation character; three steps of response were established: LPO decreasing accompanied by AOA increase; stabilization LPO <==> AOA balance; secondary LPO activation. Most early and highly amplitude decreasing of LPO were fixed in mitochondria. The rate of response have been increased on multicellular level of plants organization.