NOTE: End date changed to 04/30/2023 per NSSC (Ed., 2/1/22)

We plan to test the effect of space flight in the Exploration Mission-1 (EM-1) [now Artemis] Orion capsule on Arabidopsis thaliana seeds with enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

NOTE: End date changed to 04/30/2023 per NSSC (Ed., 2/1/22)

We plan to test the effect of space flight in the Exploration Mission-1 (EM-1) [now Artemis] Orion capsule on Arabidopsis thaliana seeds with enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

We plan to test the effect of space flight in the Exploration Mission-1 (EM-1) [now Artemis] Orion capsule on Arabidopsis thaliana seeds with enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

Life Beyond Earth: Effect of Space Flight on Seeds with Improved Nutritional Value

Principal Investigator: Federica Brandizzi, Ph.D., Michigan State University.

Participating Researchers: Evan Angelos, Ph.D. candidate, Michigan State University – Obtained PhD in Fall 2021. Joanne Thomson, Ph.D. candidate, Michigan State University.

Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival on extraterrestrial surfaces. Owing to their small volume and minimal storage requirements, seeds are the most convenient means of plant transportation in space. However, through as yet largely unknown mechanisms, space flight affects seed quality and, consequently, the vigor of seedlings. Therefore, more research is needed to fully understand seed biology responses to space flight and, ultimately, make plants a sustainable source of oxygen and food in space. To increase fundamental knowledge of plants’ responses to space flight and to facilitate germplasm selection for extended space flight and extraterrestrial colonization, this experiment will test the effect of space flight on Arabidopsis thaliana seeds enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

In our laboratory, we are studying established Arabidopsis thaliana mutants with a significantly altered BCAA biosynthetic pathway. The key enzymes in the pathway constitute a small group that includes isopropylmalate synthase 1 (IPMS1), threonine deaminase (named L-O-methylthreonine resistant 1 (OMR1 in Arabidopsis), and acetohydroxyacid synthase (AHAS). These enzymes are responsible for committed steps of BCAA biosynthesis from threonine and pyruvate. The IPMS1 mutant ipms1-1D, the AHASS small subunit 2 mutant, (ahass2-1D) and the allosteric domain mutant of OMR1 (omr1-11D) show over-accumulation of certain BCAAs compared to wild type (WT), while the levels of the other amino acids remain unchanged. Therefore, these mutants are very informative to test, specifically the effect of overabundance of certain BCAAs on growth and stress responses in a model plant species. Because space flight conditions can impoverish the nutrient reserves of seeds, we therefore hypothesize that seeds with increased levels of BCAAs will not only better endure space flight associated stress and produce healthier seedlings, but could also supply a greater proportion of mission critical dietary requirements. This information will be very helpful in selecting seed genotypes for resisting space stress and in selecting food with enhanced nutritional value to humans on extraterrestrial surfaces.

After the successful completion of the Science Verification Test (SVT), we have worked on the Experiment Verification Test (EVT) experiment using seeds from all the plants of the same genotype that were pooled to create the EVT test generation. We conducted germination and viability tests. The results of these experiments were considered within the superior category of the germination and viability success criterion. We also conducted heterotrophic assays by measuring hypocotyl length of seedlings grown in the dark, with the ImageJ software. The results were well below the required value for a superior rating in the assay execution success criterion. Finally, we performed autotrophic assays with 4-week-old plants grown in soil. We measured the average fresh weight of the excised shoots for each genotype. The results were below the required value for a superior rating in the assay execution success criterion. In each of the criteria which were relevant to the EVT experiment – Flight Environment Data Availability, Sample Integrity at Recovery, Seed Germination and Viability, and Germination, Heterotrophic, and Autotrophic Growth Assay Execution – we have achieved superior ratings.

1. For Flight Environment Data Availability – the data from the data loggers, as well as the extra thermocouples, provided excellent data over the entire course of the experiment. After reviewing the data, we have found no reason to suspect that any kind of temperature gradient within the hardware would affect the outcome of the science.

2. For Sample Integrity at Recovery – we inspected all of the returned 50ml tubes and found no evidence of broken seals or free seeds inside any of the containers.

3. For Seed Germination and Viability – all genotypes were viable at rates above the superior threshold.

4. For Germination, Heterotrophic, and Autotrophic Growth Assay Execution – all experiments were completed in a timely manner and were found to be free from any kind of microbial contamination or pest infestation.

For each experiment, the relative standard error (RSE) for all genotypes was below the 10% threshold in the superior rating. On the whole, the EVT shows the robustness and reproducibility of our experiments which will allow us to accurately assess any differences in phenotypes between flight and ground samples during the primary experiment.

In preparation for the flight, parent plants of all the genotypes are being grown in an environment-controlled growth chamber under standard Arabidopsis growth conditions (21°C under a 16h light/8h dark cycle). Plants are grown simultaneously in the same chamber and randomly distributed throughout the chamber to prevent positional bias from affecting plant health or productivity.

We plan to test the effect of space flight in the Exploration Mission-1 (EM-1) [now Artemis] Orion capsule on Arabidopsis thaliana seeds with enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

In our laboratory, we are studying established Arabidopsis thaliana mutants with a significantly altered BCAA biosynthetic pathway. The key enzymes in the pathway constitute a small group that includes isopropylmalate synthase 1 (IPMS1), threonine deaminase (named L-O-methylthreonine resistant 1 (OMR1 in Arabidopsis)), and acetohydroxyacid synthase (AHAS). These enzymes are responsible for committed steps of BCAA biosynthesis from threonine and pyruvate. The IPMS1 mutant ipms1-1D (here dubbed ipms1), the AHASS small subunit 2 mutant (ahass2-1D; herein dubbed ahass2), and the allosteric domain mutant of OMR1 (omr1-11D; herein dubbed omr1) show over-accumulation of certain BCAAs compared to WT (wild type), while the levels of the other amino acids remain unchanged. Therefore, these mutants are very informative to test specifically the effect of overabundance of certain BCAAs on growth and stress responses in a model plant species. Because spaceflight conditions can impoverish the nutrient reserves of seeds, we therefore hypothesize that seeds with increased levels of BCAAs will not only better endure spaceflight-associated stress and produce healthier seedlings, but could also supply a greater proportion of mission critical dietary requirements. This information will be very helpful in selecting seed genotypes for resisting space stress and in selecting food with enhanced nutritional value to humans on extraterrestrial surfaces.

After the successful completion of the Science Verification Test (SVT), we began preparations for the Experiment Verification Test (EVT) experiment. We performed germination and viability testing of our WT and BCAA mutants after incubation in temperatures that would have been considered on the extreme end of the possible mission temperatures. For all the genotypes tested, there were no significant differences in % germination or % viability between the experimental test temperatures (35°C, 4°C, 49°C) and the control temperature (22°C) except for a small but statistically significant difference in germination percentage between WT 22°C (99.6% germination) and WT 49°C (97.3% germination; p value= 0.02). For the EVT, either a worst-case temperature profile or average temperature profile could be used. No modifications to the success criteria are required. The data from these experiments is currently being compiled and analyzed in preparation for our EVT report which will be sent to the Kennedy Space Center Test Operations and Support Contract (KSC-TOSC) team in February.

We plan to test the effect of space flight in the Exploration Mission-1 (EM-1) [now Artemis] Orion capsule on Arabidopsis thaliana seeds with enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content.

Going forward, we are preparing for the next set of tests by determining the logistics and supplies needed for preparation and integration of our seeds into the hardware which will support multiple experiments during this mission. In our lab we are also planning our seed production schedules to guarantee the timely production of healthy seed in sufficient quantities to meet the requirements for the next set of verification test experiments, and for the flight experiment. Given the relevance of seed nutrient reserves to our hypothesis, these preparations will include production of back-up generations of seed at regular intervals to ensure that we would always have fresh stock available for flight in the unlikely event that unavoidable issues delay the launch of the mission.

To increase fundamental knowledge of plants’ responses to space flight and to facilitate germplasm selection for extended space flight and extraterrestrial colonization, in this proposal we plan to test the effect of space flight in the Exploration Mission-1 (EM-1) Orion capsule on Arabidopsis thaliana seeds enriched nutrient stores, specifically elevated levels of branched-chain amino acids (BCAAs). These essential nutrients serve as storage reserves for the seeds and seedlings. Generally, seeds with high levels of nutrients are more likely to germinate and produce healthy seedlings than seeds with low nutrient content. Additionally, development of seeds with high levels of nutrients is important to provide a compact and efficient nourishment to humans. Because space flight conditions can impoverish the nutrient reserves of seeds, we therefore hypothesize that seeds with increased levels of BCAAs will not only better endure space flight-associated stress and produce healthier seedlings, but could also supply a greater proportion of mission-critical dietary requirements.

Successful accomplishment of our research, which is grounded upon our experience with space flight with NASA Biological Research in Canisters (BRIC)-units and A. thaliana, will increase basic knowledge of the effects of space flight on fundamental plant metabolism. We expect that our results will also set important foundations for translatable principles needed to increase plant nutritional value and cultivation in space flight for the long-term colonization of extraterrestrial surfaces by humans.