PREMISE OF THE STUDY: Gravity has been a major force throughout the evolution of terrestrial organisms, and plants have developed exquisitely sensitive, regulated tropisms and growth patterns that are based on the gravity vector. The nullified gravity during spaceflight allows direct assessment of gravity roles. The microgravity environments provided by the Space Shuttle and International Space Station have made it possible to seek novel insights into gravity perception at the organismal, tissue, and cellular levels. Cell cultures of Arabidopsis thaliana perceive and respond to spaceflight, even though they lack the specialized cell structures normally associated with gravity perception in intact plants; in particular, genes for a specific subset of heat shock proteins (HSPs) and factors (HSFs) are induced. Here we ask if similar changes in HSP gene expression occur during nonspaceflight changes in gravity stimulation. METHODS: Quantitative RT-qPCR was used to evaluate mRNA levels for Hsp17.6A and Hsp101 in cell cultures exposed to four conditions: spaceflight (mission STS-131), hypergravity (centrifugation at 3 g or 16 g), sustained two-dimensional clinorotation, and transient milligravity achieved on parabolic flights. KEY RESULTS: We showed that HSP genes were induced in cells only in response to sustained clinorotation. Transient microgravity intervals in parabolic flight and various hypergravity conditions failed to induce HSP genes. CONCLUSIONS: We conclude that nondifferentiated cells do indeed sense their gravity environment and HSP genes are induced only in response to prolonged microgravity or simulated microgravity conditions. We hypothesize that HSP induction upon microgravity indicates a role for HSP-related proteins in maintaining cytoskeletal architecture and cell shape signaling.
Research Containing: Messenger
Gravistimulation changes the accumulation pattern of the CsPIN1 auxin efflux facilitator in the endodermis of the transition zone in cucumber seedlings
Cucumber (Cucumis sativus) seedlings grown in a horizontal position develop a specialized protuberance (or peg) on the lower side of the transition zone between the hypocotyl and the root. This occurs by suppressing peg formation on the upper side via a decrease in auxin resulting from a gravitational response. However, the gravity-stimulated mechanism of inducing asymmetric auxin distribution in the transition zone is poorly understood. The gravity-sensing tissue responsible for regulating auxin distribution in the transition zone is thought to be the endodermal cell. To characterize the gravity-stimulated mechanism, the auxin efflux facilitator PIN-FORMED1 (CsPIN1) in the endodermis was identified and the localization of CsPIN1 proteins during the gravimorphogenesis of cucumber seedlings was examined. Immunohistochemical analysis revealed that the accumulation pattern of CsPIN1 protein in the endodermal cells of the transition zone of cucumber seedlings grown horizontally differed from that of plants grown vertically. Gravistimulation for 30 min prompted changes in the accumulation pattern of CsPIN1 protein in the endodermis as well as the asymmetric distribution of auxin in the transition zone. Furthermore, 2,3,5-triiodobenzoic acid inhibited the differential distribution of auxin as well as changes in the accumulation pattern of CsPIN1 in the endodermis of the transition zone during gravistimulation. These results suggest that the altered pattern of CsPIN1 accumulation in the endodermis in response to gravistimulation influences lateral auxin transport through the endodermis, resulting in asymmetric auxin distribution in the transition zone.
The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells
The PICM-19 pig liver stem cell line was cultured in space for nearly 16 d on the STS-126 mission to assess the effects of spaceflight on the liver's parenchymal cells-PICM-19 cells to differentiate into either monolayers of fetal hepatocytes or 3-dimensional bile ductules (cholangiocytes). Semi-quantitative data included light microscopic assessments of final cell density, cell morphology, and response to glucagon stimulation and electron microscopic assessment of the cells' ultrastructural features and cell-to-cell connections and physical relationships. Quantitative assessments included assays of hepatocyte detoxification functions, i.e., inducible P450 activities and urea production and quantitation of the mRNA levels of several liver-related genes. Three post-passage age groups were included: 4-d-, 10-d-, and 14-d-old cultures. In comparing flight vs. ground-control cultures 17 h after the space shuttle's return to earth, no differences were found between the cultures with the exception being that some genes were differentially expressed. By light microscopy both young and older cultures, flight and ground, had grown and differentiated normally in the Opticell culture vessels. The PICM-19 cells had grown to approximately 75% confluency, had few signs of apoptosis or necrosis, and had either differentiated into monolayer patches of hepatocytes with biliary canaliculi visible between the cells or into 3-dimensional bile ductules with well-defined lumens. Ultrastructural features between flight and ground were similar with the PICM-19 cells displaying numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies, and occasional lipid vacuoles. Cell-to-cell arrangements were typical in both flight and ground-control samples; biliary canaliculi were well-formed between the PICM-19 cells, and the cells were sandwiched between the STO feeder cells. PICM-19 cells displayed inducible P450 activities. They produced urea in a glutamine-free medium and produced more urea in response to ammonia. The experiment's aim to gather preliminary data on the PICM-19 cell line's suitability as an in vitro model for assessments of liver function in microgravity was demonstrated, and differences between flight and ground-control cultures were minor.
Microgravity effects on leaf morphology, cell structure, carbon metabolism and mRNA expression of dwarf wheat
The use of higher plants as the basis for a biological life support system that regenerates the atmosphere, purifies water, and produces food has been proposed for long duration space missions. The objective of these experiments was to determine what effects microgravity (microg) had on chloroplast development, carbohydrate metabolism and gene expression in developing leaves of Triticum aestivum L. cv. USU Apogee. Gravity naive wheat plants were sampled from a series of seven 21-day experiments conducted during Increment IV of the International Space Station. These samples were fixed in either 3% glutaraldehyde or RNAlater or frozen at -25 degrees C for subsequent analysis. In addition, leaf samples were collected from 24- and 14-day-old plants during the mission that were returned to Earth for analysis. Plants grown under identical light, temperature, relative humidity, photoperiod, CO(2), and planting density were used as ground controls. At the morphological level, there was little difference in the development of cells of wheat under microg conditions. Leaves developed in mug have thinner cross-sectional area than the 1g grown plants. Ultrastructurally, the chloroplasts of microg grown plants were more ovoid than those developed at 1g, and the thylakoid membranes had a trend to greater packing density. No differences were observed in the starch, soluble sugar, or lignin content of the leaves grown in microg or 1g conditions. Furthermore, no differences in gene expression were detected leaf samples collected at microg from 24-day-old leaves, suggesting that the spaceflight environment had minimal impact on wheat metabolism.
Mechanical load induces upregulation of transcripts for a set of genes implicated in secondary wall formation in the supporting tissue of Arabidopsis thaliana
We examined the effects of mechanical load on transcripts of a set of cell wall related genes that are implicated in the formation of supporting tissues, by applying a 50 mg strip of aluminum foil to the inflorescence stem of Arabidopsis thaliana, a weight roughly half the fresh weight of the stem. Transcript levels of 12 of the 15 genes examined were increased by load application, as were the levels of some transcription factors that regulate secondary wall formation. These findings support the involvement of a load-sensing system in regulation of supporting tissue formation via transcriptional regulation of cell wall related genes.