ALBERT

Description: 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.

Description: Proper exchange of atmospheric gases is important for normal root and shoot metabolism in plants. This study was conducted to determine how restricted air supply affects foliar carbohydrates, while using the marker enzyme alcohol dehydrogenase (ADH) to report on the oxygenation status of the rootzone. Fourteen-day-old Arabidopsis thaliana (L.) Heynh. plants grown singly in 7-ml tubes containing agarified nutrient medium were placed in coupled Magenta vessels and exposed for six days to either ambient air or one of six different air/nitrogen dilutions. Redox potential of the agar medium was measured immediately after harvesting and freezing leaf tissue, and then root systems were quickly extracted from the agar and frozen for subsequent analyses. Redox potential measurements indicated that this series of gas mixtures produced a transition from hypoxia to anoxia in the root zones. Root ADH activity increased at higher rates as the redox potential neared anoxic levels. In contrast, ADH mRNA expression quickly neared its maximum as the medium became hypoxic and showed little further increase as it became anoxic. Foliar carbohydrate levels increased 1.5- to 2-fold with decreased availability of metabolic gases, with starch increasing at higher concentrations of air than soluble carbohydrate. The results serve as a model for plant performance under microgravity conditions, where absence of convective air movement prevents replenishment of metabolic gases.

Description: 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.

Description: 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.

Description: 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.

Description: The development of pollen and ovules in Arabidopsis thaliana on the space shuttle 'Endeavour' (STS-54) was investigated. Plants were grown on nutrient agar for 14 days prior to loading into closed plant growth chambers that received light and temperature control inside the Plant Growth Unit flight hardware on the shuttle middeck. After 6 days in spaceflight the plants were retrieved and immediately dissected and processed for light and electron microscope observation. Reproductive development aborted at an early stage. Pistils were collapsed and ovules inside were seen to he empty. No viable pollen was observed from STS-54 plants; young microspores were deformed and empty. At a late stage, the cytoplasm of the pollen contracted and became disorganized, but the pollen wall developed and the exine appeared normal. The tapetum in the flight flowers degenerated at early stages. Ovules from STS-54 flight plants stopped growing and the integuments and nucellus collapsed and degenerated. The megasporocytes appeared abnormal and rarely underwent meiosis. Apparently they enlarged, or occasionally produced a dyad or tetrad, to assume the form of a female gametophyte with the single nucleus located in an egglike cell that lacks a cell wall. Synergids, polar nuclei, and antipodals were not observed. The results demonstrate the types of lesions occurring in plant reproductive material under spaceflight conditions.

Description: Successful development of seeds under spaceflight conditions has been an elusive goal of numerous long-duration experiments with plants on orbital spacecraft. Because carbohydrate metabolism undergoes changes when plants are grown in microgravity, developing seed storage reserves might be detrimentally affected during spaceflight. Seed development in Arabidopsis thaliana plants that flowered during 11 d in space on shuttle mission STS-68 has been investigated in this study. Plants were grown to the rosette stage (13 d) on a nutrient agar medium on the ground and loaded into the Plant Growth Unit flight hardware 18 h prior to lift-off. Plants were retrieved 3 h after landing and siliques were immediately removed from plants. Young seeds were fixed and processed for microscopic observation. Seeds in both the ground control and flight plants are similar in their morphology and size. The oldest seeds from these plants contain completely developed embryos and seed coats. These embryos developed radicle, hypocotyl, meristematic apical tissue, and differentiated cotyledons. Protoderm, procambium, and primary ground tissue had differentiated. Reserves such as starch and protein were deposited in the embryos during tissue differentiation. The aleurone layer contains a large quantity of storage protein and starch grains. A seed coat developed from integuments of the ovule with gradual change in cell composition and cell material deposition. Carbohydrates were deposited in outer integument cells especially in the outside cell walls. Starch grains decreased in number per cell in the integument during seed coat development. All these characteristics during seed development represent normal features in the ground control plants and show that the spaceflight environment does not prevent normal development of seeds in Arabidopsis.

Description: 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.

Description: 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.

Description: Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.