Dissecting Low Atmospheric Pressure Stress: Transcriptome Responses to the Components of Hypobaria in Arabidopsis [Experiment 1]

Controlled hypobaria presents biology with an environment that is never encountered in terrestrial ecology, yet the apparent components of hypobaria are stresses typical of terrestrial ecosystems. High altitude, for example, presents terrestrial hypobaria always with hypoxia as a component stress, since the relative partial pressure of O2 is constant in the atmosphere. Laboratory-controlled hypobaria, however, allows the dissection of pressure effects away from the effects typically associated with altitude, in particular hypoxia, as the partial pressure of O2 can be varied. In this study, whole transcriptomes of plants grown in ambient (97 kPa/pO2 = 21 kPa) atmospheric conditions were compared to those of plants transferred to five different atmospheres of varying pressure and oxygen composition for 24 h: 50 kPa/pO2 = 10 kPa, 25 kPa/pO2 = 5 kPa, 50 kPa/pO2 = 21 kPa, 25 kPa/pO2 = 21 kPa, or 97 kPa/pO2 = 5 kPa. The plants exposed to these environments were 10 day old Arabidopsis seedlings grown vertically on hydrated nutrient plates. In addition, 5 day old plants were also exposed for 24 h to the 50 kPa and ambient environments to evaluate age-dependent responses. The gene expression profiles from roots and shoots showed that the hypobaric response contained more complex gene regulation than simple hypoxia, and that adding back oxygen to normoxic conditions did not completely alleviate gene expression changes in hypobaric responses. The transcriptional profiles of Arabidopsis growing in atmospheric pressures of 50 kPa or 25 kPa, either with or without supplemental oxygen, were used to evaluate the consequence of removing the hypoxic stress component from the hypobaric environment. 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 the following six treatments for 24 hours: 1) 97 kPa, 2) 50 kPa, 3) 25 kPa, 4) 50 kPa with oxygen supplemented to a partial pressure of 21 kPa (defined as 50kPa/NormOx), 5) 25 kPa with oxygen supplement to a partial pressure of 21 kPa (defined as 25kPa/NormOx) and 6) 97 kPa with reduced oxygen to a partial pressure of 5 kPa (defined as 97kPa/HypOx). At the same time, 5d plants were transferred to LPGCs and exposed to 97 kPa, 50 kPa, and 50 kPa with oxygen supplemented to a partial pressure of 21 kPa (50kPa/NormOx) for 24 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. The light, temperature and humidity remained the same as mentioned above. 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.