Performing Department
Horticultural Science
Non Technical Summary
Atmospheric pressure and composition are among the engineering variables considered in the design and construction of spaceflight vehicles and extraterrestrial habitats. Simply put, the costs of maintaining a pressure vessel at one atmosphere have been traded away throughout the history of spaceflight vehicle design and are traded away in future designs - yet the biological impacts of lower atmospheric pressure, especially on plants, is both profound and poorly understood. The objective of this proposal is to develop a refined understanding of the metabolic processes involved in plant responses and physiological adaptations to low pressure environments relevant to space exploration. The long-term goal of this line of research is a fundamental understanding of low pressure plant biology within exploration vehicles and structures, with a practical goal of producing plants that are specifically designed to thrive in low atmospheric pressures. The essential drivers of this project are the inescapable engineering limitations to producing orbital, lunar or Martian plant growth facilities that contain earth-normal atmospheric pressures, and the knowledge that plants do mount complex, and sometimes unexpected costly metabolic responses to hypobaria. These responses can also be used as a model that will allow fundamental understanding of how plants adapt to changing terrestrial environments. In the initial phases of the project, we will use the tools of functional genomics in Arabidopsis plants to examine molecular genetic responses that occur during initial and chronic exposure to atmospheres ranging from earth normal to near Martian pressures and containing various gas mixtures, especially those atmospheres that are in future vehicle designs. We will build on our previous molecular work using Arabidopsis (Paul et al. 2004). We will conduct extensive tests of a range of appropriate pressures and compositions using wild type genotypes. Then we will use select mutants and engineered changes in metabolic pathways implicated by the initial gene expression array data to test hypotheses regarding the fundamental mechanisms associated with the hypobaria response. Finally, we propose a novel use of ISS airlock facilities to investigate low pressure processes directly in a spaceflight microgravity environment, to take a first look at the emergent effects of altering atmospheric pressure within an operational vehicle in space.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Atmospheric pressure and composition are among the engineering variables considered in the design and construction of spaceflight vehicles and extraterrestrial habitats. Simply put, the costs of maintaining a pressure vessel at one atmosphere have been traded away throughout the history of spaceflight vehicle design and are traded away in future designs - yet the biological impacts of lower atmospheric pressure, especially on plants, is both profound and poorly understood. The objective of this proposal is to develop a refined understanding of the metabolic processes involved in plant responses and physiological adaptations to low pressure environments relevant to space exploration. The long-term goal of this line of research is a fundamental understanding of low pressure plant biology within exploration vehicles and structures, with a practical goal of producing plants that are specifically designed to thrive in low atmospheric pressures. The essential drivers of this project are the inescapable engineering limitations to producing orbital, lunar or Martian plant growth facilities that contain earth-normal atmospheric pressures, and the knowledge that plants do mount complex, and sometimes unexpected costly metabolic responses to hypobaria. These responses can also be used as a model that will allow fundamental understanding of how plants adapt to changing terrestrial environments. In the initial phases of the project, we will use the tools of functional genomics in Arabidopsis plants to examine molecular genetic responses that occur during initial and chronic exposure to atmospheres ranging from earth normal to near Martian pressures and containing various gas mixtures, especially those atmospheres that are in future vehicle designs. We will build on our previous molecular work using Arabidopsis (Paul et al. 2004). We will conduct extensive tests of a range of appropriate pressures and compositions using wild type genotypes. Then we will use select mutants and engineered changes in metabolic pathways implicated by the initial gene expression array data to test hypotheses regarding the fundamental mechanisms associated with the hypobaria response. Finally, we propose a novel use of ISS airlock facilities to investigate low pressure processes directly in a spaceflight microgravity environment, to take a first look at the emergent effects of altering atmospheric pressure within an operational vehicle in space.The overall goal of our program is to understand fundamental molecular biological responses to extraterrestrial and spaceflight environments. The current proposal for ground-based research leading to spaceflight addresses specifically a focus area defined by the Decadal Study: Plant and Microbial Growth Under Altered Atmospheric Pressures. The proposed research will provide fundamental insights into the biological impact of novel atmospheric environments, a focus area that is specifically identified in the Decadal Study (Decadal-Survey-Commitee 2011).
Project Methods
Aim 1 - to define the low atmospheric pressure transcriptomeThe goal for Aim 1 is to describe in detail the basic plant response to low pressure environments by using whole genome microarrays to evaluate the complete transcriptome response to each treatment. This approach will map the genes involved in the metabolic response induced by each hypobaric environment, which then sets the stage for Aim 2.General experimental setup The atmospheric treatment experiments will be conducted as triplicate sets. The chambers are connected such that three separate chambers receive the same feed and control of atmospheres. There are three sets of chambers (9 total), thus three atmospheric treatments can be conducted at any given time. The experiments will be designed to take advantage of this configuration by planning three treatments in parallel.Atmosphere profilesThe initial survey atmospheric profiles will be 97 kPa (ambient pressure in Guelph, Canada) 75 kPa, 50 kPa, 25 kPa, 10 kPa and 5 kPa; each of which will be kept at a partial pressure of CO2 of 350 ppm with the balance of nitrogen. Additional tests will be conducted with atmospheres modified with respect to oxygen: 25 kPa with O2 added to equal normoxic conditions, and 97 kPa but with a lower oxygen partial pressure to equal ppO2 at 25 kPa (5.5 % O2). These additional tests will help separate hypoxic effects from hypobaric ones, and represent important internal controls. For this survey set of experiments, plants will be exposed to the test atmospheres within the low pressure growth chambers for 24 hrs before harvest.Plant material preparation and harvests. Arabidopsis (Arabidopsis thaliana L. ecotype WS) will be grown on nutrient media agar plates held in the vertical position to encourage root growth along the aerated and exposed surface of the agar (Paul et al. 2001, Paul et al. 2004, Paul et al. 2012a) (Fig. 1).Molecular analysis overviewA major analytical readout of the responses to altered atmospheric environmental will be three measures of gene expression profiling. For systems-scale profiling, we will conduct DNA microarray and RNASeq transcriptome analyses.Developmental time course and extended exposure. A series of plates each with a different Arabidopsis age (0, 3, 5, 8, 10 day old) will be started in the chambers together and allowed to grow for 5 days. The plants will then be harvested to RNAlater as described above. These will not be used for microarray analyses, but will be reserved for targeted gene expression surveys with quantitative PCR.RNA isolation, DNA microarrays and RNASeqTotal RNA from each sample will be extracted using RNAeasy™ kits from Qiagen as described by the Qiagen kit instructions. The amount of RNA predicted from each sample is sufficient for the proposed experimental design and transcriptome analyses. Again, this technique is routine in our laboratory. The target RNAs will be labeled and prepared for hybridization according to the protocols outlined in the GeneChipâ Expression Analysis Technical Manual (Revision 1, 2001, Affymetrix, Santa Clara, Ca). (Paul et al. 2004, Paul et al. 2005b, Paul et al. 2012b).Quantitative RT-PCRSelected genes identified from the microarrays will be quantified with Taqman® Real Time Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) from Applied Biosystems (ABI) (Bustin 2000). The ABI 7500 FAST instrument will be used for the analyses. The amount of targeted message in each sample will be determined by relating the Taqman® results for the gene of interest to a standard curve (Paul et al. 2004, Paul et al. 2005b, Visscher et al. 2009, Paul et al. 2011, Paul et al. 2012b).Bioinformatics overview The Bioinformatics Division at ICBR has extensive experience in the management, annotation, and assembly of complex massively parallel sequencing projects and DNA microarray projects.Aim 2 - to develop an understanding of the metabolic pathways and signalingThe goal is to build a detailed picture of specific gene expression and possible regulatory pathways based on the information derived from the global assessment of gene expression primarily conducted in Aim 1.In addition, we will obtain or create several new GFP reporter gene lines that can be used to follow the tissue-specific development of patterns of gene expression as plants mount the stress response. We have previously used Adh::GFP (hypoxia) and Cor78::GFP (cold and drought) in this manner (Paul et al. 2004; and Figures 1 and 5) and will develop new lines as suggested by the transcriptome data.Development of transgenic GFP Biomonitors In addition to using biochemical and genomic tools, we have also worked towards developing Arabidopsis plants as biological sensors of gene expression that can be used to report changes in metabolic responses telemetrically with remote sensing technologies. Biosensor plants consist of two components: the sensor promoter that monitors the cellular physiology and stress, the reporter gene that generates a visible signal; they function as biological sensors of their environment.Promoter- sGFP(S65T) constructs will be developed through PCR amplification from Arabidopsis genomic DNA using primers to the gene promoter of interest, and be designed to amplify from about 1000bp upstream from the gene start site.Imaging and data collection of GFP reporter gene linesOver the past several years we have worked with KSC scientists and engineers to test the efficacy of several versions of the GFP imaging System (GIS), a version of which is currently housed on the International Space Station.Aim 3 - to design an ISS flight experiment that addresses low pressure biologyIn this aim we describe a clear path to hypothesis testing in spaceflight experiments on the ISS, hypotheses that directly tie our ground-based studies to the spaceflight environment. We primarily propose to use the ISS Joint Airlock 'Quest' to study the integrated effects of pressure within an active vehicle in microgravity. We propose to refine the science aspects of this Aim based on the results of Aim 1 and 2, but the basic question is the biological response to the integrated environment of low pressure in an operational microgravity vehicle.Plants in our typical 10cm Petri plate configuration would be organized into a rack and placed within and just outside the airlock, and the airlock pressure would be varied for the experiments. After exposure, the plates would be returned to the Multipurpose Work Area in the US Laboratory for immediate harvest to RNAlater in KFTs, storage within the MELFI and return to the PIs for processing and comparison to the results of Aims 1 and 2 and also to ground controls conducted within the JSC Space Station Airlock Chamber (NASA 2012b).Petri plates with plants would be located just under one of the Equipment Lock's GLAs and growth conducted in the otherwise nominal airlock environment. Use of the GLAs throughout the Station would also simplify the conduct of 'flight' full-pressure control experiments in that additional plant plates could be setup under a GLA in a location outside of the airlock.Preflight testing as well as ground controls could be conducted within the JSC Space Station Airlock Chamber (NASA 2012b). This chamber would provide the same operational interfaces as well as test pressures (e.g. 70 kPa, 10.2 psi) utilized in the proposed flight experiment.