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 within space exploration vehicles and habitats. 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 contributing to the design of plants that thrive in challenging exploration environments. The essential drivers of this project are that hypobaric environments will likely be a feature of future exploration vehicles and habitats, together with the knowledge that plants mount complex and costly metabolic responses to hypobaria. Furthermore, plants mount complex and sometimes unexpected responses to spaceflight and altered gravity environments. We hypothesize that the combination of hypobaria and microgravity will have a synergistic effect on the physiological adaptation to this complex environment, and that the patterns of gene expression will reveal strategies to both understand and help ameliorate the combined effect. These responses will also inform fundamental understanding of how plants adapt to changing terrestrial habitats facing complex and novel stressful environments.

There is a facility on the ISS that is well suited for these experiments: the Combustion Integrated Rack (CIR). The CIR has a pressure vessel that can be programmed telemetrically to the gas composition and pressures relevant to our study. There is also a CIR ground unit that can be similarly programmed for parallel ground controls, thereby enabling the precise dissection of the effects of the orbital environment on plant biology within the pressure vessel.

The proposed research will provide fundamental insights into the biological impact of novel atmospheric environments, a focus area that is itself specifically identified in the Decadal Study. (Ed. Note. The National Academies of Sciences, Engineering, and Medicine Decadal Survey on Biological and Physical Sciences Research in Space 2023-2032). More importantly, this Hypobaric Plant Biology in Space Exploration study seeks to fundamentally examine combined spaceflight effects in order to develop an understanding of emergent response properties that are not predicable from an examination of the individual responses.

Pyruvate Decarboxylase 1 Knockout Seed Line

The pyruvate decarboxylase 1 knockout, pdc1, or SALK_090204c line has a T-DNA insertion within the pyruvate decarboxylase 1 gene resulting in a knockout. Germplasm of this line was received from the Arabidopsis Biological Resource Center (ABRC), grown, and genotyped in-house. Additional quantitative reverse transcription polymerase chain reaction (qRT-PCR) verification was done to verify knockout of the PDC1 gene. Both a wildtype and the pdc1 line were analyzed in both untreated and cold treated states to examine levels of expression. The knockout of the PDC1 gene was confirmed with the knockout line showing no induction and maintaining very little detectable transcripts and the wildtype displayed an approximate 7 times increase in expression of the PDC1 gene.

pOMG1::GFP Reporter Line

The pOMG1::GFP reporter line is undergoing plant screening with the goal to identify individuals homozygous for the insertion and that respond well to induction. OMG1 is known to be induced by crimping, this line is screened via crimping with a hemostat or forceps. The leaf is then examined for green fluorescent protein (GFP) expression approximately 45 minutes later. Individuals were selected and bulked for the flight experiment.

pRD29::GFP Reporter Line

The pRD29::GFP reporter line was tested using a 48 hour cold treatment at 4 degrees Celsius. Individuals were selected and bulked for the flight experiment.

Ground and Flight Hardware Development

Hypobaric Pressure Chambers

To begin testing of transgenic reporter lines and other variables, low pressure chambers are needed. The current hypobaric (low pressure) chamber system consists of 3 cylindrical chambers, a KNF vacuum controller, 2 atmospheric pressure sensors, and a monitoring computer. The chambers are interconnected with a braided vinyl tubing, with additional reinforcement of standard tube clamps at the connections. Additionally, the chambers can be managed offline independently without disturbing the other active chambers by being connected to the primary line via quick-disconnect fittings. To monitor the chambers, 3 independent monitoring systems are used, the KNF’s pressure control settings, an MSR sensor, and a Dracal USB atmospheric pressure monitor. Code has also been written to both automatically monitor and send alerts when the pressure is outside a given range and respond to pressure read out requests sent via email. Overall, the pressure chamber can maintain a monitored low-pressure environment (as low as 30 kPa) for indefinite periods of time, but current experiments have run from a few hours to up to 10 days.

Keyence Petri Plate Adapter

While the Keyence BZ-X800 has many readily available stages and inserts for use, they are adapted primarily for cell cultures and not petri plates. A microgravity environment poses even greater issues with this due to petri plate movement. To resolve these issues, we have designed, tested, 3D printed, and created a final 3D design of a Keyence petri plate adapter.

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 within space exploration vehicles and habitats. 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 contributing to the design of plants that thrive in challenging exploration environments. The essential drivers of this project are that hypobaric environments will likely be a feature of future exploration vehicles and habitats, together with the knowledge that plants mount complex and costly metabolic responses to hypobaria. Furthermore, plants mount complex and sometimes unexpected responses to spaceflight and altered gravity environments. We hypothesize that the combination of hypobaria and microgravity will have a synergistic effect on the physiological adaptation to this complex environment, and that the patterns of gene expression will reveal strategies to both understand and help ameliorate the combined effect. These responses will also inform fundamental understanding of how plants adapt to changing terrestrial habitats facing complex and novel stressful environments.

There is a facility on the ISS that is well suited for these experiments: the Combustion Integrated Rack (CIR). The CIR has a pressure vessel that can be programmed telemetrically to the gas composition and pressures relevant to our study. There is also a CIR ground unit that can be similarly programmed for parallel ground controls, thereby enabling the precise dissection of the effects of the orbital environment on plant biology within the pressure vessel.

The proposed research will provide fundamental insights into the biological impact of novel atmospheric environments, a focus area that is itself specifically identified in the Decadal Study. (Ed. Note. The National Academies of Sciences, Engineering, and Medicine Decadal Survey on Biological and Physical Sciences Research in Space 2023-2032). More importantly, this Hypobaric Plant Biology in Space Exploration study seeks to fundamentally examine combined spaceflight effects in order to develop an understanding of emergent response properties that are not predicable from an examination of the individual responses.

Testing Short-duration hypobaric treatments have been conducted. These treatments are conducted using clear hypobaric chambers retrofitted with a MSR Electronics data logger, to track barometric pressure, and a vacuum pump to draw out the gases from the chamber. Treatments typically last for 12-24 hours and are subjected to varying reduced pressures from 10-80kPA. These are general atmosphere reductions, not controlled gases. The purpose of these treatments has been to examine expression of the reporter lines in a hypobaric environment. Further analysis of the pdc1 knockout line using the hypobaric chambers, followed by expression verification through qRT-PCR, is also currently underway.

Hardware Progress The hypobaric experiment, which has been designated APEX-11 (Advanced Plant Experiments), is scheduled to use the CIR (Combustion Integrated Rack) facility and the VEGGIE growth chamber. The plants will grow for 10 days in the NASA Vegetable Production System (Veggie growth chamber) using a still-under-development plant growth rack. The Veggie rack will be designed for 40 plates, which will allow for a shortened experimental procedure process. There is also currently a separate rack in development for hypobaric treatments inside the CIR. In April 2023, Dr. Paul traveled to NASA Glenn Research Center outside of Cleveland, OH to work on a plan to integrate science into the CIR. Dr. Paul traveled with several plates to serve as mockup plates that will grow the plants for APEX-11. These plates were used to test the prototype rack. Plates were mounted on the rack and loaded into the CIR for design tests.

Future Plan Current plans call for Science Verification Test (SVT) to being in Q4 2023, with Experiment Verification Test (EVT) and Flight operations in 2024. SVT stage of operation is ready to move forward on the biology side; however, hardware operations are still in planning stages.

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 within space exploration vehicles and habitats. 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 contributing to the design of plants that thrive in challenging exploration environments. The essential drivers of this project are that hypobaric environments will likely be a feature of future exploration vehicles and habitats, together with the knowledge that plants mount complex and costly metabolic responses to hypobaria. Furthermore, plants mount complex and sometimes unexpected responses to spaceflight and altered gravity environments. We hypothesize that the combination of hypobaria and microgravity will have a synergistic effect on the physiological adaptation to this complex environment, and that the patterns of gene expression will reveal strategies to both understand and help ameliorate the combined effect. These responses will also inform fundamental understanding of how plants adapt to changing terrestrial habitats facing complex and novel stressful environments.

There is a facility on the ISS that is well suited for these experiments: the Combustion Integrated Rack (CIR). The CIR has a pressure vessel that can be programmed telemetrically to the gas composition and pressures relevant to our study. There is also a CIR ground unit that can be similarly programmed for parallel ground controls, thereby enabling the precise dissection of the effects of the orbital environment on plant biology within the pressure vessel.

The proposed research will provide fundamental insights into the biological impact of novel atmospheric environments, a focus area that is itself specifically identified in the Decadal Study. (Ed. Note. The National Academies of Sciences, Engineering, and Medicine Decadal Survey on Biological and Physical Sciences Research in Space 2023-2032). More importantly, this Hypobaric Plant Biology in Space Exploration study seeks to fundamentally examine combined spaceflight effects in order to develop an understanding of emergent response properties that are not predicable from an examination of the individual responses.

Two genotypes of Arabidopsis (Arabidopsis thaliana L.) will be used: wild ecotype Columbia-0 (Col-0) and Col-0 deficient in a gene sensitive to low pressure environments. The gene of interest is highly induced in response to hypoxia in terrestrial environments and is also induced by spaceflight. These two seed lines will be used for the bulk of the biochemical work done, such as transcriptomics, on the upcoming flight experiment. The experiment also calls for four reporter gene lines for an imaging plate to be flown along side the wild type (WT) and knockout plant lines.

These seed lines are currently in development for upcoming testing and flight experiments.

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 within space exploration vehicles and habitats. 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 contributing to the design of plants that thrive in challenging exploration environments. The essential drivers of this project are that hypobaric environments will likely be a feature of future exploration vehicles and habitats, together with the knowledge that plants mount complex and costly metabolic responses to hypobaria. Furthermore, plants mount complex and sometimes unexpected responses to spaceflight and altered gravity environments. We hypothesize that the combination of hypobaria and microgravity will have a synergistic effect on the physiological adaptation to this complex environment, and that the patterns of gene expression will reveal strategies to both understand and help ameliorate the combined effect. These responses will also inform fundamental understanding of how plants adapt to changing terrestrial habitats facing complex and novel stressful environments.

There is a facility on the ISS that is well suited for these experiments: the Combustion Integrated Rack (CIR). The CIR has a pressure vessel that can be programmed telemetrically to the gas composition and pressures relevant to our study. There is also a CIR ground unit that can be similarly programmed for parallel ground controls, thereby enabling the precise dissection of the effects of the orbital environment on plant biology within the pressure vessel.

The proposed research will provide fundamental insights into the biological impact of novel atmospheric environments, a focus area that is itself specifically identified in the Decadal Study. (Ed. Note. The National Academies of Sciences, Engineering, and Medicine Decadal Survey on Biological and Physical Sciences Research in Space 2023-2032). More importantly, this Hypobaric Plant Biology in Space Exploration study seeks to fundamentally examine combined spaceflight effects in order to develop an understanding of emergent response properties that are not predicable from an examination of the individual responses.