Dynamics of storage reserve deposition during Brassica rapa L. pollen and seed development in microgravity.

Pollen and seeds share a developmental sequence characterized by intense metabolic activity during reserve deposition before drying to a cryptobiotic form. Neither pollen nor seed development has been well studied in the absence of gravity, despite the importance of these structures in supporting future long-duration manned habitation away from Earth. Using immature seeds (3-15 d postpollination) of Brassica rapa L. cv. Astroplants produced on the STS-87 flight of the space shuttle Columbia, we compared the progress of storage reserve deposition in cotyledon cells during early stages of seed development. Brassica pollen development was studied in flowers produced on plants grown entirely in microgravity on the Mir space station and fixed while on orbit. Cytochemical localization of storage reserves showed differences in starch accumulation between spaceflight and ground control plants in interior layers of the developing seed coat as early as 9 d after pollination. At this age, the embryo is in the cotyledon elongation stage, and there are numerous starch grains in the cotyledon cells in both flight and ground control seeds. In the spaceflight seeds, starch was retained after this stage, while starch grains decreased in size in the ground control seeds. Large and well-developed protein bodies were observed in cotyledon cells of ground control seeds at 15 d postpollination, but their development was delayed in the seeds produced during spaceflight. Like the developing cotyledonary tissues, cells of the anther wall and filaments from the spaceflight plants contained numerous large starch grains, while these were rarely seen in the ground controls. The tapetum remained swollen and persisted to a later developmental stage in the spaceflight plants than in the ground controls, even though most pollen grains appeared normal. These developmental markers indicate that Brassica seeds and pollen produced in microgravity were physiologically younger than those produced in 1 g. We hypothesize that microgravity limits mixing of the gaseous microenvironments inside the closed tissues and that the resulting gas composition surrounding the seeds and pollen retards their development.

Spaceflight hardware for conducting plant growth experiments in space: the early years 1960-2000.

The best strategy for supporting long-duration space missions is believed to be bioregenerative life support systems (BLSS). An integral part of a BLSS is a chamber supporting the growth of higher plants that would provide food, water, and atmosphere regeneration for the human crew. Such a chamber will have to be a complete plant growth system, capable of providing lighting, water, and nutrients to plants in microgravity. Other capabilities include temperature, humidity, and atmospheric gas composition controls. Many spaceflight experiments to date have utilized incomplete growth systems (typically having a hydration system but lacking lighting) to study tropic and metabolic changes in germinating seedlings and young plants. American, European, and Russian scientists have also developed a number of small complete plant growth systems for use in spaceflight research. Currently we are entering a new era of experimentation and hardware development as a result of long-term spaceflight opportunities available on the International Space Station. This is already impacting development of plant growth hardware. To take full advantage of these new opportunities and construct innovative systems, we must understand the results of past spaceflight experiments and the basic capabilities of the diverse plant growth systems that were used to conduct these experiments. The objective of this paper is to describe the most influential pieces of plant growth hardware that have been used for the purpose of conducting scientific experiments during the first 40 years of research.

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh.

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.

Evidence of root zone hypoxia in Brassica rapa L. grown in microgravity.

A series of experiments was conducted aboard the U.S. space shuttle and the Mir space station to evaluate microgravity-induced root zone hypoxia in rapid-cycling Brassica (Brassica rapa L.), using both root and foliar indicators of low-oxygen stress to the root zone. Root systems from two groups of plants 15 and 30 d after planting, grown in a phenolic foam nutrient delivery system on the shuttle (STS-87), were harvested and fixed for microscopy or frozen for enzyme assays immediately postflight or following a ground-based control. Activities of fermentative enzymes were measured as indicators of root zone hypoxia and metabolism. Following 16 d of microgravity, ADH (alcohol dehydrogenase) activity was increased in the spaceflight roots 47% and 475% in the 15-d-old and 30-d-old plants, respectively, relative to the ground control. Cytochemical localization showed ADH activity in only the root tips of the space-grown plants. Shoots from plants that were grown from seed in flight in a particulate medium on the Mir station were harvested at 13 d after planting and quick-frozen and stored in flight in a gaseous nitrogen freezer or chemically fixed in flight for subsequent microscopy. When compared to material from a high-fidelity ground control, concentrations of shoot sucrose and total soluble carbohydrate were significantly greater in the spaceflight treatment according to enzymatic carbohydrate analysis. Stereological analysis of micrographs of sections from leaf and cotyledon tissue fixed in flight and compared with ground controls indicated no changes in the volume of protoplast, cell wall, and intercellular space in parenchyma cells. Within the protoplasm, the volume occupied by starch was threefold higher in the spaceflight than in the ground control, with a concomitant decrease in vacuolar volume in the spaceflight treatment. Both induction of fermentative enzyme activity in roots and accumulation of carbohydrates in foliage have been repeatedly shown to occur in response to root zone oxygen deprivation. These results indicate that root zone hypoxia is a persistent challenge in spaceflight plant growth experiments and may be caused by microgravity-induced changes in fluid and gas distribution.

Influence of atmospheric oxygen on leaf structure and starch deposition in Arabidopsis thaliana.

Plant culture in oxygen concentrations below ambient is known to stimulate vegetative growth, but apart from reports on increased leaf number and weight, little is known about development at subambient oxygen concentrations. Arabidopsis thaliana (L.) Heynh. (cv. Columbia) plants were grown full term in pre-mixed atmospheres with oxygen partial pressures of 2.5, 5.1, 10.1, 16.2, and 21.3 kPa O2, 0.035 kPa CO2 and the balance nitrogen under continuous light. Fully expanded leaves were harvested and processed for light and transmission electron microscopy or for starch quantification. Growth in subambient oxygen concentrations caused changes in leaf anatomy (increased thickness, stomatal density and starch content) that have also been described for plants grown under carbon dioxide enrichment. However, at the lowest oxygen treatment (2.5 kPa), developmental changes occurred that could not be explained by changes in carbon budget caused by suppressed photorespiration, resulting in very thick leaves and a dwarf morphology. This study establishes the leaf parameters that change during growth under low O2, and identifies the lower concentration at which O2 limitation on transport and biosynthetic pathways detrimentally affects leaf development. Grant numbers: NAG5-3756, NAG2-1020, NAG2-1375.

Reproduction during spaceflight by plants in the family Brassicaceae.

NASA: Researchers report on studies of reproduction in Arabidopsis thaliana in space during during the Chromex-03 on STS-54, Chromex-04 on STS-51, and Chromex-05 on STS-68 missions. The obstacles to seed formation were related to carbon dioxide levels. Other experiments examined in flight pollination and seed production in Brassica rapa during parabolic flight, a 4-1/2 month stay on Mir, and on STS-87. During the Mir experiment, Brassica seeds were harvested from seeds sown in flight. The second generation seeds grew to produce new seeds that contained more starch and less protein and lipid when compared to ground control seeds.^ieng

Pollination and embryo development in Brassica rapa L. in microgravity.

Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in 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.

A ground-based comparison of nutrient delivery technologies originally developed for growing plants in the spaceflight environment.

A ground-based comparison of plant nutrient delivery systems that have been developed for microgravity application was conducted for dwarf wheat (Triticum aestivum L. 'Yecora Rojo') and rapid-cycling brassica (Brassica rapa L. CrGC#1-33) plants. These experiments offer insight into nutrient and oxygen delivery concerns for greenhouse crop production systems. The experiments were completed over a 12-day period to simulate a typical space shuttle-based spaceflight experiment. The plant materials, grown either using the porous-tube nutrient delivery system, the phenolic foam support system, or a solidified agar nutrient medium, were compared by plant-growth analysis, root zone morphological measurements, elemental composition analysis, and alcohol dehydrogenase enzyme activity assay. The results of these analyses indicate that the porous tube plant nutrient delivery and the phenolic foam systems maintain plant growth at a higher level than the solidified agar gel medium system. Root zone oxygenation problems associated with the agar system were manifested through biochemical and morphological responses. The porous tube nutrient delivery system outperformed the other two systems on the basis of plant growth analysis parameters and physiological indicators of root zone aeration. This information is applicable to the current crop production techniques used in greenhouse-controlled environments.

Realizing the potential of rapid-cycling Brassica as a model system for use in plant biology research.

Rapid-cycling Brassica populations were initially developed as a model for probing the genetic basis of plant disease. Paul Williams and co-workers selected accessions of the six main species for short time to flower and rapid seed maturation. Over multiple generations of breeding and selection, rapid-cycling populations of each of the six species were developed. Because of their close relationship with economically important Brassica species, rapid-cycling Brassica populations, especially those of B. rapa (RCBr) and B. oleracea, have seen wide application in plant and crop physiology investigations. Adding to the popularity of these small, short-lived plants for research applications is their extensive use in K-12 education and outreach.

ASTROCULTURE (TM) root metabolism and cytochemical analysis.

Physiology of the root system is dependent upon oxygen availability and tissue respiration. During hypoxia nutrient and water acquisition may be inhibited, thus affecting the overall biochemical and physiological status of the plant. For the Astroculture (TM) plant growth hardware, the availability of oxygen in the root zone was measured by examining the changes in alcohol dehydrogenase (ADH) activity within the root tissue. ADH activity is a sensitive biochemical indicator of hypoxic conditions in plants and was measured in both spaceflight and control roots. In addition to the biochemical enzyme assays, localization of ADH in the root tissue was examined cytochemically. The results of these analyses showed that ADH activity increased significantly as a result of spaceflight exposure. Enzyme activity increased 248% to 304% in dwarf wheat when compared with the ground controls and Brassica showed increases between 334% and 579% when compared with day zero controls. Cytochemical staining revealed no differences in ADH tissue localization in any of the dwarf wheat treatments. These results show the importance of considering root system oxygenation in designing and building nutrient delivery hardware for spaceflight plant cultivation and confirm previous reports of an ADH response associated with spaceflight exposure.

Influence of microgravity on ultrastructure and storage reserves in seeds of Brassica rapa L.

Successful plant reproduction under spaceflight conditions has been problematic in the past. During a 122 d opportunity on the Mir space station, full life cycles of Brassica rapa L. were completed in microgravity in a series of three experiments in the Svet greenhouse. Ultrastructural and cytochemical analyses of storage reserves in mature dry seeds produced in these experiments were compared with those of seeds produced during a high-fidelity ground control. Additional analyses were performed on developing Brassica embryos, 15 d post pollination, which were produced during a separate experiment on the Shuttle (STS-87). Seeds produced on Mir had less than 20% of the cotyledon cell number found in seeds harvested from the ground control. Cytochemical localization of storage reserves in mature cotyledons showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in ground control seeds. Protein bodies in mature cotyledons produced in space were 44% smaller than those in the ground control seeds. Fifteen days after pollination, cotyledon cells from mature embryos formed in space had large numbers of starch grains, and protein bodies were absent, while in developing ground control seeds at the same stage, protein bodies had already formed and fewer starch grains were evident. These data suggest that both the late stage of seed development and maturation are changed in Brassica by growth in a microgravity environment. While gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.

Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development.

Growth of Arabidopsis thaliana (L.) Heynh. in decreasing oxygen partial pressures revealed a linear decrease in seed production below 15 kPa, with a complete absence of seed production at 2.5 kPa oxygen. This control of plant reproduction by oxygen had previously been attributed to an oxygen effect on the partitioning between vegetative and reproductive growth. However, plants grown in a series of decreasing oxygen concentrations produced progressively smaller embryos that had stopped developing at progressively younger stages, suggesting instead that their growth is limited by oxygen. Internal oxygen concentrations of buds, pistils, and developing siliques of Brassica rapa L. and siliques of Arabidopsis were measured using a small-diameter glass electrode that was moved into the structures using a micromanipulator. Oxygen partial pressures were found to be lowest in the developing perianth (11.1 kPa) and pistils (15.2 kPa) of the unopened buds. Pollination reduced oxygen concentration inside the pistils by 3 kPa after just 24 h. Inside Brassica silique locules, partial pressures of oxygen averaged 12.2 kPa in darkness, and increased linearly with increasing light levels to 16.2 kPa. Measurements inside Arabidopsis siliques averaged 6.1 kPa in the dark and rose to 12.2 kPa with light. Hypoxia in these microenvironments is postulated to be the point of control of plant reproduction by oxygen.

The tropic response of plant roots to oxygen: oxytropism in Pisum sativum L.

Plant roots are known to orient growth through the soil by gravitropism, hydrotropism, and thigmotropism. Recent observations of plant roots that developed in a microgravity environment in space suggested that plant roots may also orient their growth toward oxygen (oxytropism). Using garden pea (Pisum sativum L. cv. Weibul's Apollo) and an agravitropic mutant (cv. Ageotropum), root oxytropism was studied in the controlled environment of a microrhizotron. A series of channels in the microrhizotron allowed establishment of an oxygen gradient of 0.8 mmol mol-1 mm-1. Curvature of seedling roots was determined prior to freezing the roots for subsequent spectrophotometric determinations of alcohol dehydrogenase activity. Oxytropic curvature was observed all along the gradient in both cultivars of pea. The normal gravitropic cultivar showed a maximal curvature of 45 degrees after 48 h, while the agravitropic mutant curved to 90 degrees. In each cultivar, the amount of curvature declined as the oxygen concentration decreased, and was linearly related to the root elongation rate. Since oxytropic curvature occurred in roots exposed to oxygen concentrations that were not low enough to induce the hypoxically responsive protein alcohol dehydrogenase, we suspect that the oxygen sensor associated with oxytropism does not control the induction of hypoxic metabolism. Our results indicate that oxygen can play a critical role in determining root orientation as well as impacting root metabolic status. Oxytropism allows roots to avoid oxygen-deprived soil strata and may also be the basis of an auto-avoidance mechanism, decreasing the competition between roots for water and nutrients as well as oxygen.

Changes in Arabidopsis leaf ultrastructure, chlorophyll and carbohydrate content during spaceflight depend on ventilation.

Leaf structure and function under spaceflight conditions have received little study despite their important implications for biological life support systems using plants. Previous reports described disruption of the membrane apparatus for photosynthesis and a general decrease in carbohydrate content in foliage. During a series of three short-duration experiments (Chromex-03, -04, -05) on the US space shuttle (STS-54, STS-51, STS-68), we examined Arabidopsis thaliana leaves. The plants were at the rosette stage at the time of loading onto the space shuttle, and received the same light, temperature, carbon dioxide and humidity regimes in the orbiter as in ground controls. The experiments differed according to the regime provided in the headspace around the plants: this was either sealed (on mission STS-54); sealed with high levels of carbon dioxide (on mission STS-51) or vented to the cabin air through a filtration system (on mission STS-68). Immediately post-flight, leaf materials were fixed for microscopy or frozen in liquid nitrogen for subsequent analyses of chlorophyll and foliar carbohydrates. At the ultrastructural level, no aberrations in membrane structure were observed in any of the experiments. When air-flow was provided, plastids developed large starch grains in both spaceflight and ground controls. In the experiments with sealed chambers, spaceflight plants differed from ground controls with regard to measured concentrations of carbohydrate and chlorophyll, but the addition of airflow eliminated these differences. The results point to the crucial importance of consideration of the foliage microenvironment when spaceflight effects on leaf structure and metabolism are studied.

Waterlogging effects on growth and yield components in late-planted soybean.

A major agronomic problem in the southeastern USA is low yield of late-planted soybean [Glycine max (L.) Merr.]. This problem is aggravated by the adverse effect of waterlogging on crop growth. Our objectives were to identify soybean growth stages sensitive to waterlogging; identify yield components and physiological parameters explaining yield losses induced by waterlogging; and determine the extent of yield losses induced by waterlogging under natural field conditions. Greenhouse and field studies were conducted during 1993 and 1994 near Baton Rouge, LA, (30 degrees N Lat) on a Commerce silt loam. Waterlogging tolerance was assessed in cultivar Centennial (Maturity Group VI) at three vegetative and five reproductive growth stages by maintaining the water level at the soil surface in a greenhouse study. Using the same cultivar, we evaluated the effect of drainage in the field for late-planted soybean. Rain episodes determined the timing of waterlogging; redox potential and oxygen concentration of the soil were used to quantify the intensity of waterlogging stress. Results of the greenhouse study indicated that the early vegetative period (V2) and the early reproductive stages (R1, R3, and R5) were most sensitive to waterlogging. Three to 5 cm of rain per day falling on poorly drained soil was sufficient to reduce crop growth rate, resulting in a yield decline from 2453 to 1550 kg ha-1. Yield loss in both field and greenhouse studies was induced primarily by decreased pod production resulting from fewer pods per reproductive node. In conclusion, waterlogging was determined to be an important stress for late-planted soybean in high rainfall areas such as the Gulf Coast Region.

Control of seed development in Arabidopsis thaliana by atmospheric oxygen.

Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5.1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2.5, 5.1, 10.1, 16.2 and 21.3 kPa O2, 0.035 kPa CO2 and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O2 treatments below 16.2 kPa, and seeds from plants grown in 2.5 kPa O2 did not germinate at all. Fewer than 25% of the seeds from plants grown in 5.1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5.1 kPa O2, at around the curled cotyledon stage in 10.1 kPa O2, and at the premature stage in 16.2 kPa O2. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2.5 kPa O2. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis.

Plant reproduction in spaceflight environments.

Because plant reproduction is a complex developmental process there are many possible sites of perturbation by the unusual environments of orbital spacecraft. Previous long-duration experiments on Soviet platforms shared features of slowed development through the vegetative stage of plant growth and aborted reproductive function. Our goal has been to understand how special features of the spaceflight environment impact physiological function and reproductive development. In a series of short-duration experiments in the Shuttle mid-deck we studied early reproductive development in Arabidopsis thaliana. Pollen and ovule development aborted at an early stage in the first experiment on STS-54 which utilized closed plant growth chambers. Post-flight analysis suggested that the plants may have been carbon dioxide limited. Subsequent experiments utilized carbon dioxide enrichment (on STS-51) and cabin air flow-through with an air exchange system (on STS-68). Both modifications allowed pollen and ovule development to occur normally on orbit, and full reproductive development up to the stage of an immature seed occurred on STS-68. However, analysis of plant roots from these experiments demonstrated a limitation in rootzone aeration in the spaceflight material that was not mitigated by these procedures. In the future, additional resources (crew time, upgraded flight hardware, and special platforms) will invite more elaborate, long-duration experimentation. On the ISS, a variable speed centrifuge and upgraded plant habitats will permit detailed experiments on the role of gravity in shaping the plant micro-environment, and what influence this plays during reproduction.

Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana.

Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.

Plant reproduction during spaceflight: importance of the gaseous environment.

Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.