Mary E. Musgrave

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

Abstract. 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 gravitys 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.

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Abstract. Potassium antimonate was used to locate Ca2+ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion.

Modification of reproductive development in Arabidopsis thaliana under spaceflight conditions

Reproductive development in Arabidopsis thaliana (L.) Heynh. cv. Columbia plants was investigated under spaceflight conditions on shuttle mission STS-51. Plants launched just prior to initiation of the reproductive phase developed flowers and siliques during the 10-d flight. Approximately 500 flowers were produced in total by the 12 plants in both the ground control and spaceflight material, and there was no significant difference in the number of flowers in each size class. The flower buds and siliques of the spaceflight plants were not morphologically different from the ground controls. Pollen viability tests immediately post-flight using fluorescein diacetate indicated that about 35% of the pollen was viable in the spaceflight material. Light-microscopy observations on this material showed that the female gametophytes also had developed normally to maturity. However, siliques from the spaceflight plants contained empty, shrunken ovules, and no evidence of pollen transfer to stigmatic papillae was found by light microscopy immediately post-flight or by scanning electron microscopy on fixed material. Short stamen length and indehiscent anthers were observed in the spaceflight material, and a film-like substance inside the anther that connected to the tapetum appeared to restrict the release of pollen from the anthers. These observations indicate that given appropriate growing conditions, early reproductive development in A. thaliana can occur normally under spaceflight conditions. On STS-51, reproductive development aborted due to obstacles in pollination or fertilization.

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.

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.

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

Abstract. 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. Weibuls 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° after 48 h, while the agravitropic mutant curved to 90°. 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.

Gravity control of growth form in Brassica rapa and Arabidopsis thaliana (Brassicaceae): consequences for secondary metabolism.

How gravity influences the growth form and flavor components of plants is of interest to the space program because plants could be used for food and life support during prolonged missions away from the planet, where that constant feature of Earths environment does not prevail. We used plant growth hardware from prior experiments on the space shuttle to grow Brassica rapa and Arabidopsis thaliana plants during 16-d or 11-d hypergravity treatments on large-diameter centrifuge rotors. Both species showed radical changes in growth form, becoming more prostrate with increasing g-loads (2-g and 4-g). In Brassica, height decreased and stems thickened in a linear relationship with increasing g-load. Glucosinolates, secondary compounds that contribute flavor to Brassica, decreased by 140% over the range of micro to 4-g, while the structural secondary compound, lignin, remained constant at ∼15% (w/w) cell wall dry mass. Stem thickening at 4-g was associated with substantial increases in cell size (47%, 226%, and 33% for pith, cortex, and vascular tissue), rather than any change in cell number. The results, which demonstrate the profound effect of gravity on plant growth form and secondary metabolism, are discussed in the context of similar thigmostresses such as touch and wind.

Oxygen insensitivity of photosynthesis by waterlogged Apios americana

Abstract Apios americana, a tuber-producing viny legume, occurs naturally in areas which experience episodic soil waterlogging. In a 2-year study, plants were grown under well-drained or waterlogged soil conditions. Tuber production did occur in the waterlogged situation, but yield was substantially depressed. Specific leaf weight increased by a factor of 40% compared to the well-drained controls. Likewise, increases in soluble carbohydrate and starch were observed in leaf tissue under waterlogged conditions. Because of the increase in carbohydrate content in the foliage with waterlogging, plants were probed for feedback limitations on photosynthesis. Net assimilation rates of single leaves measured under ambient conditions were not different between well-drained and waterlogged treatments. However, oxygen insensitivity of photosynthesis was observed under waterlogged conditions, particularly in one genetic line which had high mortality in the waterlogged treatment. Well-drained plants demonstrated similar stimulations of photosynthesis by 2% oxygen regardless of genetic background. No interaction was observed between drainage and oxygen content of the assay gas in determining stomatal conductance values. Oxygen insensitivity of photosynthesis may be an indication of poor adaptation to rootzone waterlogging.

Reproduction on orbit by plants in the Brassicaceae family

Previous studies on growth and development during spaceflight had indicated that the transition from vegetative to reproductive growth was particularly difficult for plants. Our objective has been to study how the spaceflight environment impacts the different steps in plant reproduction. This goal has been pursued in two general ways: by using plants that had been pre-grown to the flowering stage on earth, and by using plants that developed completely on orbit. Our objectives have been met by a combination of experiments that required essentially no crew time on orbit, and those that required an extensive commitment of crew time. The plants chosen for the studies were closely related members of the family Brassicaceae: Arabidopsis thaliana and Brassica rapa. In a series of short-duration experiments with Arabidopsis on the space shuttle we found that depletion of carbon dioxide in closed chambers resulted in aborted development of both the male and female reproductive apparatus in microgravity. Normal dev...