Extreme hypobaria is a novel abiotic stress that is outside the evolutionary experience of terrestrial plants. In natural environments, the practical limit of atmospheric pressure experienced by higher plants is about 50 kPa or ~.5 atmospheres; a limit that is primarily imposed by the combined stresses inherent to high altitude conditions of terrestrial mountains. However, in highly controlled chambers and within extra-terrestrial greenhouses the atmospheric pressure component can be isolated from other high altitude stresses such as temperature, desiccation, and even hypoxia. In addition, hypobaria can be carried to extremes beyond what is possible in terrestrial biomes, and explored as a single variable in the examination of plant responses to novel stress. Previous studies have shown that plants adjust to hypobaric stress by differentially expressing suites of genes in unique combinations that are not equal to the dissected components of hypobaric stress (such as hypoxia and desiccation). Here we examine the organ-specific progression of transcriptional strategies for physiological adaptation to hypobaric stress over time. An abrupt transition from a near-sea level pressure of 97 kPa to only 5 kPa is accompanied by the differential expression of hundreds of genes. However, the transcriptomic reaction to hypobaric conditions lying between these two extremes reveals complex, organ-specific responses that vary over a time course of hypobaric exposure, and that are also not linear with respect to a simple gradient of severity. It is also clear that plants adjust over time such that the gene expression patterns that are initially elicited to cope with hypobaria are mediated as plants adjust their metabolism to this environment. The patterns of genome-wide changes in gene expression across a gradient of atmospheric pressures, and over a time course of several days allows for the development of theories of how plant metabolisms may be adapting to changes in atmospheric pressures.
Overall design: The transcriptional profiles of Arabidopsis growing in atmospheric pressures of 75 kPa, 50 kPa, 25 kPa, 10 kPa or 5 kPa were used to evaluate the consequence of the hypobaric environment. In the first experimnent the 10 day old plants were transferred to The Low Pressure Growth Chambers (LPGC) (the Controlled Environment Systems Research Facility (CESRF) at University of Guelph, Ontario, Canada) and exposed to 97 kPa, 75 kPa, 50 kPa, 25 kPa, 10 kPa, 5 kPA for 24 hours, respectively. In the second experiment the 10 days old plants were transferred to the LPGCs and were exposed to an atmosphere of 10 kPa or 97 kPa. The 97 kPa atmosphere was composed of a partial pressure of 21 kPa Oxygen, 0.05 kPa Carbon Dioxide and a balance of Nitrogen. The 10 kPa samples were composed of a partial pressure of 2.1 kPa Oxygen, 0.05 kPa Carbon Dioxide and a balance of Nitrogen. The samples were harvested at: 1 hour, 3 hours, 6 hours, 12 hours, 48 hours and 72 hours. The carbon dioxide was held constant at a partial pressure of 0.05 kPa in all treatments. Nitrogen was used as a balance of remaining gas for oxygen treatments. Each atmospheric treatment was replicated in three different chambers, and each chamber held 10 individual plates comprised of 12 plants each. At the completion of each atmospheric treatment, plants were harvested from media surface directly to RNAlater (Ambion). For each treatment, there were three chambers each containing 10 plates of plants in total. Approximately 12 plants from each plate were harvested to a separate tube and were immediately stored as previously described (Paul and Ferl, 2011). One tube was selected from each LPGC replicate, for a total of three tubes per treatment group. The plants were dissected into shoots (entire aerial portion of the plant including hypocotyl) and roots and the total RNA was extracted and subjected to the The Affymetrix GeneChip® Arabidopsis ATH1 Genome Arrays.
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