Patterns of Arabidopsis gene expression in the face of hypobaric stress [Experiment 2]

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. 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.

极端低气压（extreme hypobaria）是一种全新的非生物胁迫（abiotic stress），超出了陆生植物（terrestrial plants）的演化经历范围。在自然环境中，高等植物（higher plants）所经历的大气压力（atmospheric pressure）实际下限约为50 kPa或约0.5个标准大气压；该下限主要由陆地山脉高海拔环境固有的多重胁迫共同施加。然而，在高度可控的培养箱（controlled chambers）以及外星温室（extra-terrestrial greenhouses）中，大气压力这一变量可与其他高海拔胁迫（如温度胁迫、脱水（desiccation）胁迫乃至低氧（hypoxia）胁迫）分离开来。此外，低气压可被调控至陆生生物群落中无法实现的极端水平，可作为单一变量用于探究植物对新型胁迫的响应。
既往研究表明，植物通过以独特组合方式差异表达多组基因来适应低气压胁迫，这种基因表达模式与低气压胁迫的拆解组分（如低氧与脱水胁迫）并不等同。本研究旨在探究随时间推移，植物适应低气压胁迫的器官特异性转录调控策略（transcriptional strategies）的动态进程。
将植物从近海平面的97 kPa气压环境骤降至仅5 kPa时，会伴随数百个基因的差异表达。但介于这两个极端之间的低气压环境下的转录组响应（transcriptomic response）则呈现出复杂的器官特异性动态变化：其随低气压暴露时长而改变，且与简单的严重程度梯度并不呈线性关系。此外，植物会随时间进行适应性调整，最初为应对低气压而诱导的基因表达模式，会随着植物将代谢机制适配至该环境而发生改变。
全基因组基因表达模式随大气压力梯度以及数天的时间进程发生的变化，可为阐释植物代谢如何适应大气压力改变提供理论依据。本研究利用在75 kPa、50 kPa、25 kPa、10 kPa或5 kPa大气压力下生长的拟南芥（Arabidopsis）的转录谱，评估低气压环境的影响。
第一个实验中，将10日龄的拟南芥幼苗转移至低压力生长箱（Low Pressure Growth Chambers, LPGC，即加拿大安大略省圭尔夫大学受控环境系统研究设施（Controlled Environment Systems Research Facility, CESRF）），分别在97 kPa、75 kPa、50 kPa、25 kPa、10 kPa、5 kPa的气压环境下暴露24小时。
第二个实验中，将10日龄的拟南芥幼苗转移至LPGC，分别暴露于10 kPa或97 kPa的大气环境。其中97 kPa大气环境的组分为：氧气分压21 kPa、二氧化碳分压0.05 kPa，其余气体为氮气；10 kPa大气环境的组分为：氧气分压2.1 kPa、二氧化碳分压0.05 kPa，其余气体为氮气。
样本分别在处理后1小时、3小时、6小时、12小时、48小时和72小时收获。所有处理组的二氧化碳分压均维持在0.05 kPa，剩余气体以氮气补充以维持设定的氧气分压。每个大气处理组设置3个独立培养箱作为生物学重复，每个培养箱内放置10个培养板，每板含12株拟南芥。
每个大气处理结束后，直接从培养基表面将植株收获至RNAlater（Ambion）中。每个处理组的每个培养箱中，从每块培养板选取约12株植株至单独的离心管中，按照此前的方法（Paul和Ferl, 2011）立即进行保存。从每个LPGC重复样本中选取1支离心管，每个处理组共计3支离心管。将植株拆解为地上部分（包含下胚轴的全部地上组织）与根系，提取总RNA后使用Affymetrix GeneChip®拟南芥ATH1全基因组芯片（Affymetrix GeneChip® Arabidopsis ATH1 Genome Arrays）进行转录组检测。