Developmental, nutritional and hormonal anomalies of weightlessness-grown wheat.

The behavior of water in weightlessness, as occurs in orbiting spacecraft, presents multiple challenges for plant growth. Soils remain saturated, impeding aeration, and leaf surfaces remain wet, impeding gas exchange. Herein we report developmental and biochemical anomalies of "Super Dwarf" wheat (Triticum aestivum L.) grown aboard Space Station Mir during the 1996-97 "Greenhouse 2" experiment. Leaves of Mir-grown wheat were hyperhydric, senesced precociously and accumulated aromatic and branched-chain amino acids typical of tissues experiencing oxidative stress. The highest levels of stress-specific amino acids occurred in precociously-senescing leaves. Our results suggest that the leaf ventilation system of the Svet Greenhouse failed to remove sufficient boundary layer water, thus leading to poor gas exchange and onset of oxidative stress. As oxidative stress in plants has been observed in recent space-flight experiments, we recommend that percentage water content in apoplast free-spaces of leaves be used to evaluate leaf ventilation effectiveness. Mir-grown plants also tillered excessively. Crowns and culms of these plants contained low levels of abscisic acid but high levels of cytokinins. High ethylene levels may have suppressed abscisic acid synthesis, thus permitting cytokinins to accumulate and tillering to occur.

[Performance characteristics of root zone moisture and water potential sensors for greenhouses in the conditions of extended space flight].

The investigation was performed using greenhouse Lada in the Russian segment of the International space station (ISS RS) as part of space experiment Plants-2 during ISS missions 5 through to 22. A set of 6 point moisture sensors embedded in the root zone (turface particles of 1-2 mm in diam.) and 4 tensiometers inside root modules (RM) were used to monitor moisture content and water potential in the root zone. The purpose was to verify functionality and to test performance of the sensors in the spacefight environment. It was shown that with the average RZ moisture content of 80% the measurement error of the sensors do not exceed ± 1.5%. Dynamic analysis of the tensiometers measurements attests that error in water potential measurements does not exceed ± 111 Pa.

Farming in space: environmental and biophysical concerns.

The colonization of space will depend on our ability to routinely provide for the metabolic needs (oxygen, water, and food) of a crew with minimal re-supply from Earth. On Earth, these functions are facilitated by the cultivation of plant crops, thus it is important to develop plant-based food production systems to sustain the presence of mankind in space. Farming practices on earth have evolved for thousands of years to meet both the demands of an ever-increasing population and the availability of scarce resources, and now these practices must adapt to accommodate the effects of global warming. Similar challenges are expected when earth-based agricultural practices are adapted for space-based agriculture. A key variable in space is gravity; planets (e.g. Mars, 1/3 g) and moons (e.g. Earth's moon, 1/6 g) differ from spacecraft orbiting the Earth (e.g. Space stations) or orbital transfer vehicles that are subject to microgravity. The movement of heat, water vapor, CO2 and O2 between plant surfaces and their environment is also affected by gravity. In microgravity, these processes may also be affected by reduced mass transport and thicker boundary layers around plant organs caused by the absence of buoyancy dependent convective transport. Future space farmers will have to adapt their practices to accommodate microgravity, high and low extremes in ambient temperatures, reduced atmospheric pressures, atmospheres containing high volatile organic carbon contents, and elevated to super-elevated CO2 concentrations. Farming in space must also be carried out within power-, volume-, and mass-limited life support systems and must share resources with manned crews. Improved lighting and sensor technologies will have to be developed and tested for use in space. These developments should also help make crop production in terrestrial controlled environments (plant growth chambers and greenhouses) more efficient and, therefore, make these alternative agricultural systems more economically feasible food production systems.

Plant growth during the Greenhouse II experiment on the Mir orbital station.

We carried out three experiments with Super Dwarf wheat in the Bulgarian/Russian growth chamber Svet (0.1 m2 growing area) on the Space Station Mir. This paper mostly describes the first of these NASA-supported trials, began on Aug. 13, 1995. Plants were sampled five times and harvested on Nov. 9 after 90 days. Equipment failures led to low irradiance (3, then 4 of 6 lamp sets failed), instances of high temperatures (ca. 37 degrees C), and sometimes excessive substrate moisture. Although plants grew for the 90 d, no wheat heads were produced. Considering the low light levels, plants were surprisingly green, but of course biomass production was low. Plants were highly disoriented (low light, mirror walls?). Fixed and dried samples and the root module were returned on the U.S. Shuttle Atlantis on Nov. 20, 1995. Samples of the substrate, a nutrient-charged zeolite called Balkanine, were taken from the root module, carefully examined for roots, weighed, dried, and reweighed. The Svet control unit and the light bank were shipped to Moscow. An experiment validation test (EVT) of plant growth and experimental procedures, carried out in Moscow, was highly successful. Equipment built in Utah to measure CO2, H2O vapor, irradiance, air and leaf (IR) temperature, O2, pressure, and substrate moisture worked well in the EVT and in space. After this manuscript was first prepared, plants were grown in Mir with a new light bank and controller for 123 d in late 1996 and 39 days in 1996/1997. Plants grew exceptionally well with higher biomass production than in any previous space experiment, but the ca. 280 wheat heads that were produced in 1996 contained no seeds. Ethylene in the cabin atmosphere was responsible.

[The evaluation of the error of the thermal pulse technique used to measure moisture content of root substrates in space greenhouse]. / Otsenka pogreshnosti teploimpul'snogo metoda izmereniia vlagosoderzhaniia korneobitaemykh sred dlia kosmicheskikh oranzherei.

Point thermopulse probes were used to monitor moisture level in the root substrates during cultivation of higher plants in a space greenhouse. Investigated were performance data of the thermopulse moisture probe in integration with the space greenhouse. It was shown that within the substrate moisture range from 20 up to 100% of the full saturation the technique error does not exceed 1.5%. The thermopulse technique bears much promise for metrologic monitoring of the root substrate moisture content in space greenhouses no matter water and air supply technology.

Comparative floral development of Mir-grown and ethylene-treated, earth-grown Super Dwarf wheat.

To study plant growth in microgravity, we grew Super Dwarf wheat (Triticum aestivum L.) in the Svet growth chamber onboard the orbiting Russian space station, Mir, and in identical ground control units at the Institute of BioMedical Problems in Moscow, Russia. Seedling emergence was 56% and 73% in the two root-module compartments on Mir and 75% and 90% on earth. Growth was vigorous (produced ca. 1 kg dry mass), and individual plants produced 5 to 8 tillers on Mir compared with 3 to 5 on earth-grown controls. Upon harvest in space and return to earth, however, all inflorescences of the flight-grown plants were sterile. To ascertain if Super Dwarf wheat responded to the 1.1 to 1.7 micromoles mol-1 atmospheric levels of ethylene measured on the Mir prior to and during flowering, plants on earth were exposed to 0, 1, 3, 10, and 20 micromoles mol-1 of ethylene gas and 1200 micromoles mol-1 CO2 from 7 d after emergence to maturity. As in our Mir wheat, plant height, awn length, and the flag leaf were significantly shorter in the ethylene-exposed plants than in controls; inflorescences also exhibited 100% sterility. Scanning-electron-microscopic (SEM) examination of florets from Mir-grown and ethylene-treated, earth-grown plants showed that development ceased prior to anthesis, and the anthers did not dehisce. Laser scanning confocal microscopic (LSCM) examination of pollen grains from Mir and ethylene-treated plants on earth exhibited zero, one, and occasionally two, but rarely three nuclei; pollen produced in the absence of ethylene was always trinucleate, the normal condition. The scarcity of trinucleate pollen, abrupt cessation of floret development prior to anthesis, and excess tillering in wheat plants on Mir and in ethylene-containing atmospheres on earth build a strong case for the ethylene on Mir as the agent for the induced male sterility and other symptoms, rather than microgravity.

Gravity independence of seed-to-seed cycling in Brassica rapa.

Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.

[Growth of wheat from seed-to-seed in space flight]. / Vyrashchivanie pshenitsy "ot semeni do semeni" v usloviiakh kosmicheskogo poleta.

In our earlier space experiment with super dwarf wheat we found the spikes developed in space to be barren. The cause of the full crop sterility was sensitivity of this wheat species to the ethylene concentration of 0.3-0.8 mg/m3 during the experiment. The follow-up ground experiments were made to identify species of dwarf wheat that could be raised in space greenhouse Svet and are distinguished by partial tolerance of their reproductive organs to elevated ethylene in air. The choice fell on the USU-Apogee cultivar specially developed for planting in growth chambers as an integral part of various bioregenerative life support systems, including the space ones. An experiment with wheat Apogee was performed in greenhouse Svet on board MIR. The period of the full crop vegetation cycle was not significantly altered under the spaceflight conditions. The experiment yielded 508 seeds from 12 plants, i.e. by 38% less than in laboratory experiments and by 69% more as compared with results of growing crops in ethylene-contaminated atmosphere (1 mg/m3). Mass of the space seeds was low if compared with the laboratory crops. This was the first time when the feasibility of gathering seeds from wheat that had passed the whole vegetation cycle in space flight was demonstrated. The experiment will give mightly impetus to the advancement of research on space biological LSS and gravitational biology.

Canopy photosynthesis and transpiration in microgravity: gas exchange measurements aboard Mir.

The SVET Greenhouse on-board the Orbital Station Mir was used to measure canopy photosynthesis and transpiration rates for the first time in space. During the Greenhouse IIB experiment on Mir (June-January 1997), carbon and water vapor fluxes from two wheat (cv. Superdwarf) canopies were measured using the US developed Gas Exchange Measurement System (GEMS). Gas analyzers capable of resolving CO2 concentration differences of 5 micromoles mol-1 against a background of 0.9% CO2, are necessary to measure photosynthetic and respiratory rates on Mir. The ability of the GEMS gas analyzers to measure these CO2 concentration differences was determined during extensive ground calibrations. Similarly, the sensitivity of the analyzers to water vapor was sufficient to accurately measure canopy evapotranspiration. Evapotranspiration, which accounted for over 90% of the water added to the root zone, was estimated using gas exchange and used to estimate substrate moisture content. This paper presents canopy photosynthesis and transpiration data during the peak vegetative phase of development in microgravity.

Analysis of the spaceflight effects on growth and development of Super Dwarf wheat grown on the Space Station Mir.

The hypothesis being tested is that Super Dwarf wheat, Triticum aestivum L., plants in the Svet Greenhouse onboard the Russian Space Station Mir will complete a life cycle in spaceflight, providing that the environmental conditions necessary for adequate growth on Earth are supplied. Twenty six seeds of wheat were planted in each of 2 rows of 2 root compartments for a total of 104 seeds in Svet. Germination rate at 7 d was 56 and 73% on Mir and 75 and 90% in ground-based controls. Plants were grown throughout the whole cycle of ontogenesis (123 d) with samples gathered at different times to validate the morphological and reproductive stages of the plants. Young plants showed vigorous early seedling growth, with large biomass production, including the formation of 280 floral spikes. Upon return to Earth, comparative analyses showed that the number of tillers and flowers per spikelet were 63.2% and 40% greater, respectively, in Mir-grown plants than in the controls. By contrast, the stem length (52.4%), spike mass (49.2%) and length (23.1%), awn length (75.7%), number of spikelets per spike (42.8%) and number of seeds per spike (100% sterile) from Mir-grown plants were substantially less than the controls. Distribution of moisture and roots throughout the substrate was very good. All florets on Mir-grown spikes ceased development at the same stage of ontogeny. Lack of caryopses formation was attributed to male sterility occurring at different stages of staminal development. Anthers failed to dehisce and pollen grains were smaller and shriveled compared to the controls, suggesting a chronic stress had occurred in the Svet growth chamber. Recent ground-based studies indicated that ethylene, which was measured at 0.3 to 1.8 mg kg-1 in the Mir, almost certainly could have induced male sterility in the wheat plants grown on the Mir.

Silica deposition on the leaves of Mir- and Earth-grown Super Dwarf wheat.

Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) microanalysis were used to investigate the nature of crystals deposited on leaves of Mir- and Earth-grown Super Dwarf wheat (Triticum aestivum L.) plants. Leaves from these plants exhibited dense and uniformly distributed crystals on leaf abaxial surfaces when viewed by SEM. Young leaves showed that crystals initially accumulated around the stomata on the adaxial surface, but became more dense and uniformly distributed as the leaves aged. EDX microanalyses of the Balkanine (a nutrient charged clinoptilolite zeolite) medium in which the wheat plants were grown showed an elemental pattern similar to that observed on the wheat leaves. The absence of N and P in the Balkanine suggests that they were completely utilized by the plants. Only Si and O were evident in the drying agent, Sorb-it-Silica (TM), and perhaps could have accounted for some of the Si observed on the plant tissue. Grant numbers: NCC2-831, 84322-4810

Microgravity effects on water supply and substrate properties in porous matrix root support systems.

The control of water content and water movement in granular substrate-based plant root systems in microgravity is a complex problem. Improper water and oxygen delivery to plant roots has delayed studies of the effects of microgravity on plant development and the use of plants in physical and mental life support systems. Our international effort (USA, Russia and Bulgaria) has upgraded the plant growth facilities on the Mir Orbital Station (OS) and used them to study the full life cycle of plants. The Bulgarian-Russian-developed Svet Space Greenhouse (SG) system was upgraded on the Mir OS in 1996. The US developed Gas Exchange Measurement System (GEMS) greatly extends the range of environmental parameters monitored. The Svet-GEMS complex was used to grow a fully developed wheat crop during 1996. The growth rate and development of these plants compared well with earth grown plants indicating that the root zone water and oxygen stresses that have limited plant development in previous long-duration experiments have been overcome. However, management of the root environment during this experiment involved several significant changes in control settings as the relationship between the water delivery system, water status sensors, and the substrate changed during the growth cycles.

[Some peculiar features of liquid supply to the root medium of plants growing in microgravity]. / Nekotorye osobennosti obespecheniia korneobitaemoi sredy pri kul'tivirovanii rastenii v usloviiakh nevesomosti.

Sixteen point probes monitored moisture level in the root medium of the wheat plants grown in greenhouse SVET on the MIR/NASA space science program. The article outlines types of water migration in the absence of gravity. Hydrophysical characteristics of perspective root media have been explored. Results of the water supply monitoring and control in the course of experiment are reported. The authors put forward porous root media to facilitate water migration and aeration.

[Evaluation of super dwarf wheat growth and development in greenhouse "Svet" during cultivation in inhabited pressurized chamber]. / Issledovanie rosta i razvitiia superkarlikovoi pshenitsy v oranzheree "Svet" pri kul'tivirovanii v obitaemom germoob''ekte.

Goals of the 3-month experiment GREENHOUSE using the equipment of greenhouse SVET (ECO-PSY-95) were to feature growth and development of wheat through the entire cycle of ontogeny under the maximally mimicked MIR environment, and to try out the procedures and timeline of space experiment GREENHOUSE-2 as a part of the fundamental biology investigations within the MIR/NASA space science program. Irradiation intensity (PAR) was 65 W/m2 and 38 W/m2 in the experiment and laboratory control, respectively. Values of other environmental parameters were MIR average (18-25 degrees C, relative air humidity in the interval between 40% and 75%, total gas pressure of about 660 to 860 mm Hg, partial oxygen pressure within the range from 140 to 200 mm Hg, partial carbon dioxide pressure up to 7 mm Hg). Experimental results showed that wheat cultivation in inhabited chamber under a modified lighting unit providing greater irradiation of the crop area produced more plant mass although seed production dropped. Low grain content in ears could be the aftermath of the gaseous trace contaminants in the chamber atmosphere.

Six-month space greenhouse experiments--a step to creation of future biological life support systems.

SVET Space Greenhouse (SG)--the first automated facility for growing of higher plants in microgravity was designed in the eighty years to be used for the future BLSS. The first successful experiment with vegetables was carried out in 1990 on the MIR Space Station (SS). The experiments in SVET SG were resumed in 1995, when an American Gas Exchange Measurement System (GEMS) was added. A three-month wheat experiment was carried out as part of MIR-SHUTTLE'95 program. SVET-2 SG Bulgarian equipment of a new generation with optimised characteristics was developed (financed by NASA). The new SVET-GEMS equipment was launched on board the MIR SS and a successful six-month experiments for growing up of two crops of wheat were conducted in 1996 - 97 as part of MIR-NASA-3 program. The first of these "Greenhouse" experiments (123 days) with the goal to grow wheat through a complete life cycle is described. Nearly 300 heads developed but no seeds were produced. A second crop of wheat was planted and after 42 days the plants were frozen for biochemical investigations. The main environmental parameters during the six-month experiments in SVET (substrate moisture and lighting period) are given. The results and the contribution to BLSS are discussed.

The Spacelab-Mir-1 "Greenhouse-2" experiment.

The Spacelab-Mir-1 (SLM-1) mission is the first docking of the Space Shuttle Atlantis (STS-71) with the Orbital Station Mir in June 1995. The SLM-1 "Greenhouse-2" experiment will utilize the Russian-Bulgarian-developed plant growth unit (Svet). "Greenhouse-2" will include two plantings (1) designed to test the capability of Svet to grow a crop of Superdwarf wheat from seed to seed, and (2) to provide green plant material for post-flight analysis. Protocols, procedures, and equipment for the experiment have been developed by the US-Russian science team. "Greenhouse-2" will also provide the first orbital test of a new Svet Instrumentation System (SIS) developed by Utah State University to provide near real time data on plant environmental parameters and gas-exchange rates. SIS supplements the Svet control and monitoring system with additional sensors for substrate moisture, air temperature, IR leaf temperature, light, oxygen, pressure, humidity, and carbon-dioxide. SIS provides the capability to monitor canopy transpiration and net assimilation of the plants growing in each vegetation unit (root zone) by enclosing the canopy in separate, retractable, ventilated leaf chambers. Six times during the seed-to-seed experiment, plant samples will be collected, leaf area measured, and plant parts fixed and/or dried for ground analysis. A second planting initiated 30 days before the arrival of a U.S. Shuttle [originally planned to be STS-71] is designed to provide green material at the vegetative development stage for ground analysis. [As this paper is being edited, the experiment has been delayed until after the arrival of STS-71.]

Growing super-dwarf wheat in Svet on Mir.

In cooperation with Russian colleagues, we will carry out three experiments with a super-dwarf cultivar of wheat in the plant growth chamber Svet on the Russian Space Station Mir: an early test of the root module and other instruments (July-August, 1995), a seed-to-seed experiment (1996), and a third planting that will be harvested after about 35 days, frozen, and returned to Earth for chemical analysis (1996). Plants will be photographed, sampled,and chemically fixed at intervals during the first two plantings. Instrumentation has been constructed to measure CO2, water vapor, air temperatures, infrared leaf temperatures, oxygen, pressure, irradiance levels, and moisture in the root module substrate (Balkanine). The primary objective of this equipment is to allow controllers to eliminate plant environmental stresses, leading to vigorous plant growth in microgravity. We are testing sampling and analysis techniques and growing plants in ground versions of Svet in Moscow and in Svet mockups in Utah: 12 chambers in two temperature-controlled rooms, with two compartments in each chamber (two temperatures, four CO2 concentrations, and three photoperiods in a current experiment).

Influence of elevated carbon dioxide on water relations of soybeans.

Soybean (Glycine max L. Merrill cv ;Bragg') plants were grown in pots at six elevated atmospheric CO(2) concentrations and two watering regimes in open top field chambers to characterize leaf xylem potential, stomatal resistance and conductance, transpiration, and carbohydrate contents of the leaves in response to CO(2) enrichment and water stress conditions. Groups of plants at each CO(2) concentration were subjected to water stress by withholding irrigation for 4 days during the pod-filling stage.Under well watered conditions, the stomatal conductance of the plants decreased with increasing CO(2) concentration. Therefore, although leaf area per plant was greater in the high CO(2) treatments, the rate of water loss per plant decreased with CO(2) enrichment. After 4 days without irrigation, plants in lower CO(2) treatments showed greater leaf tissue damage, lower leaf water potential, and higher stomatal resistance than high CO(2) plants. Stomatal closure occurred at lower leaf water potentials for the low CO(2) grown plants than the high CO(2) grown plants. Significantly greater starch concentrations were found in leaves of high CO(2) plants, and the reductions in leaf starch and increases in soluble sugars due to water stress were greater for low CO(2) plants. The results showed that even though greater growth was observed at high atmospheric CO(2) concentrations, lower rates of water use delayed and, thereby, prevented the onset of severe water stress under conditions of low moisture availability.

Response of agronomic and forest species to elevated atmospheric carbon dioxide.

The effects of atmospheric carbon dioxide on corn, soybeans, loblolly pine, and sweetgum were studied in the field during a growing season. The plants were exposed to a range of concentrations of carbon dioxide day and night in open-topped, flow-through chambers. At a mean daytime carbon dioxide concentration of 910 parts per million, increases in total biomass ranged from 157 to 186 percent of the control values. Seed yield and wood volume increased and there were changes in plant anatomy and form. Net photosynthesis increased with increasing carbon dioxide concentration in soybeans and sweetgum, but was unaffected in corn. Water use efficiency also increased in corn, soybeans, and sweetgum.