This website could be intermittent Saturday Mar 30, 2024 starting 7PM until next day 11AM Eastern Time due to server/facility maintenance. We apologize for any inconvenience.

 

Menu

 

The NASA Task Book
Advanced Search     

Project Title:  Dissecting Beneficial Plant-Microbe Interactions and Their Efficacy in the ISS Spaceflight Environment, a Model Study Reduce
Images: icon  Fiscal Year: FY 2024 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Microbiology   | Plant Biology  
Start Date: 01/01/2020  
End Date: 12/31/2024  
Task Last Updated: 11/28/2023 
Download report in PDF pdf
Principal Investigator/Affiliation:   Lewis, Norman G Ph.D. / Washington State University 
Address:  Institute of Biological Chemistry 
299 Clark Hall 
Pullman , WA 99164-6340 
Email: lewisn@wsu.edu 
Phone: 509-335-2682  
Congressional District:
Web: http://ibc.wsu.edu/research-faculty/lewis/  
Organization Type: UNIVERSITY 
Organization Name: Washington State University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Davin, Laurence  Ph.D. Washington State University, Pullman 
Kahn, Michael  Ph.D. Washington State University, Pullman 
Project Information: Grant/Contract No. 80NSSC19K1484 
Responsible Center: NASA KSC 
Grant Monitor: Levine, Howard  
Center Contact: 321-861-3502 
howard.g.levine@nasa.gov 
Unique ID: 12525 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1484 
Project Type: FLIGHT 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
(3) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: End date changed to 12/31/2024 per NSSC information (Ed, 3/12/24)

NOTE: End date changed to 12/31/2023 per NSSC information (Ed, 1/15/23)

Task Description: Significance of objectives to NASA and this Solicitation: Deep space exploration or extraterrestrial colonization (e.g., Moon or Mars) will require the ability to sustainably produce plants for human/animal consumption, as well as providing aesthetic benefits of plant life to future crews and personnel in extra-terrestrial colonies. One key challenge in spaceflight/microgravity is in overcoming long-standing difficulties in efficaciously providing water and nutrients to germinating and maturing plants. Another important research challenge that has seen little attention is in productively exploiting beneficial plant-microbe interactions in spaceflight/microgravity, particularly for nitrogen (N) fixation. When both challenges are resolved for optimal, productive, and efficacious plant growth in space, this will provide the exciting opportunity to recycle organically bound carbon (C) and N that was sequestered in these plants. Through subsequent recycling of those organics (e.g., derived from human and animal consumption waste and from unused plant parts), this will help enable sustainable plant growth over multiple generations. Another benefit of studying beneficial plant microbe interactions is at the fundamental science level, i.e., by gaining much improved understanding of how the spaceflight/microgravity environment affects this important physiological process.

Central objectives of proposed research: Complementary purposes of our 2 Specific Aims are to initially dissect, understand, and optimize plant growth/development in spaceflight/microgravity via exploiting beneficial plant-microbe interactions. Then to ultimately recycle organic C and N from them suitable for subsequent multiple plant generations. To do this, we will use model Medicago plant species (e.g., alfalfa), and its beneficial bacterial symbiont, which together can potentially displace the need for N-containing fertilizer in spaceflight/microgravity.

Specific Aims:

1. Comprehensively compare and contrast efficacy of beneficial symbiotic plant-microbe interactions between Medicago and Sinorhizobium growing on the International Space Station (ISS) and on Earth (NASA Kennedy Space Center/KSC), including understanding changes occurring at the molecular level;

2. Compare and contrast ease of biodegradation of the ISS and Earth grown Medicago plant material, in order to assess whether there are any differences in the ability to recycle C, N, etc. for multiple generations of plant growth/development.

Justification for this work is threefold: The first is to demonstrate that beneficial plant microbe interactions during N-fixation can be efficaciously achieved in spaceflight/microgravity. The second is to gain a fundamental understanding of spaceflight/microgravity environment effects on these beneficial plant microbe interactions, and their potential usage for deep space exploration and colonization. The third is to demonstrate that organic C and N can be facilely recycled to support multiple generations of plant growth in space.

Methods/techniques: During growth, FluorPen and plant size measurements will be carried out to assess comparative N-fixation efficacy for each condition, both on the ISS and on Earth. Tissues (leaf, stem, and root) from the ISS and Earth control will be collected after ca. 6-8 weeks growth, frozen (-160°C). They will be subjected to transcriptomic and metabolomic (including amino acid) analyses; the microbiomes present in aerial/underground tissues will be determined. The multi-omics approaches employed are as for our Arabidopsis study.

Medicago plant material, from the ISS and ground control, will also be subjected to biodegradation to establish whether there are any differences in N-mineralization (for recycling) in spaceflight/microgravity or ground control tissues.

Research Impact/Earth Benefits: Among the benefits on Earth envisaged: improving our knowledge of N-fixing process and the symbiosis between Medicago and Sinorhizobium, and determining optimal lignin contents for space and Earth will be very instructive, as will the recycling C/N capabilities for both wild type and genetically modified plant lines. Demonstrating this in space is also a very effective means of demonstrating to aspiring young scientists (including Middle and High School students) and others of the importance of plant life, of N-fixation, and of C/N recycling in a sustainable manner.

Task Progress & Bibliography Information FY2024 
Task Progress: 1. Progress on Ground Verification (GVT) Test Preparations and Execution

The GVT used 2 NASA Vegetable Production System (Veggie) units, each housing twelve APEX Growth Chambers (AGCs). Twelve had an alfalfa reduced-lignin line and its corresponding control (VEGGIE 1; lignin experiment); whereas the other twelve had M. sativa v. Ladak (VEGGIE 2; symbiosis experiment).

For VEGGIE #1 (lignin experiment), the overall goal is to compare and contrast the ease of biodegradation of alfalfa grown on the International Space Station (ISS) and Earth, including low-lignin alfalfa plants. This is to assess whether there are differences in the ability to recycle C, N, etc., for multiple generations of plant growth/development. Alfalfa was grown with added nitrogen.

For VEGGIE #2 (symbiosis experiment), the hypothesis is that rhizobia will be able to generate good plant growth and root nodulation in plant growth medium lacking an added nitrogen source. There are 3 experimental configurations to test this hypothesis:

• Rhizobia (Sinorhizobium meliloti) added: This configuration should generate good plant growth and root nodulation in a plant growth medium lacking added nitrogen. • No rhizobia or added nitrogen source in plant growth medium: In this configuration, the plants should be stunted because they lack access to a usable nitrogen source. • No rhizobia, but added nitrogen source. This configuration is to test the growth of the plants when supplied with nitrogen-containing fertilizer.

1.1. GVT-1

For the first GVT, the Principal Investigator (PI) team used the Plant Growth Systems (PGSs), previously named Apex Growth Chambers (AGCs).

Members of the PI team travelled to NASA Kennedy Space Center (KSC) on May 20, 2023 and 24 PGSs were assembled. A Magenta box was next placed on the top of each PGS.

The GVT was initiated May 23, 2023, by addition of nutrient solution (125 ml) to each PGS. Each PGS was weighed before and after addition of nutrient solution. This was done at this stage in order to estimate nutrient solution loss (due to plant growth and evaporation) over time.

As the nutrient solution was dispensed, the PGSs were next placed in both Veggie systems in the ISS Environmental Simulator (ISSES) chamber. Light intensity and photoperiod were set. Temperature, relative humidity (RH), and CO2 level settings used in the ISSES chamber were those of “ISS average”.

Temperature, RH, and CO2 levels were recorded daily by KSC personnel, with averages of each being 22.6 °C, 39%, and 2,610 ppm, respectively.

In addition, two HOBO Data Loggers (one in each Veggie system) were programmed to measure and record temperature, RH, and light at 15-minute intervals during plant growth. Pictures were taken on days 6, 8, 10, 13 and every 3 days thereafter until harvest.

Eight days after initiation (DAI) of the experiment, the top Magenta box on each PGS was removed. Additional water was provided at day 10, and every 3 days thereafter until harvest, with weights again taken before and after adding water. Germination rate (10 DAI) and number of plants that grew in each PGS were recorded.

GVT-1 did not fully meet our success criteria, due to germination issues.

1.2. GVT-2

GVT-2 employed fresh seed stocks and was initiated October 5, 2023 and carried out as described above. From a biomass production perspective, GVT-2 appeared to be fully successful in meeting all Success Criteria. Plant tissues are currently under analyses.

2. Lignin-reduced Alfalfa Lines

As described in the previous Progress Report, application of CRISPR/Cas9 was carried out to generate lignin-reduced alfalfa lines. The CRISPR/Cas9 gene-editing approach was selected to disable arogenate dehydratase (ADT) genes, potentially allowing for multiple gene knock-out targets in a single experiment. To date, over 100 CRISPR/Cas9 transgenic plants have been generated.

Bibliography: Description: (Last Updated: 11/28/2023) 

Show Cumulative Bibliography
 
Abstracts for Journals and Proceedings Lewis NG, Davin LB, Costa MA, Moinuddin SGA, Hanschen ER, Starkenburg SR, Koehler SI, Winnacott BJ, Hixson KK, Lipton MS, Hanson DT, Turpin MM, Monje O, Richards JT, Dufour N, Levine HG. "Integrated multi-omics analyses of lignin-reduced Arabidopsis lines on International Space Station." XXXI International Conference on Polyphenols - ICP2023, Nantes, France, July 3-6, 2023.

Abstracts. XXXI International Conference on Polyphenols - ICP2023, Nantes, France, July 3-6, 2023. , Jul-2023

Abstracts for Journals and Proceedings Lewis NG, Costa MA, Moinuddin SGA, Mortimer MW, Kahn ML, Davin LB. "Ground-based alfalfa-Sinorhizobium symbiosis in APEX systems." 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023.

Abstracts. 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. , Nov-2023

Articles in Peer-reviewed Journals Barker R, Kruse CPS, Johnson C, Saravia-Butler A, Fogle H, Chang HS, Trane RM, Kinscherf N, Villacampa A, Manzano A, Herranz R, Davin LB, Lewis NG, Perera I, Wolverton C, Gupta P, Jaiswal P, Reinsch SS, Wyatt S, Gilroy S. "Meta-analysis of the space flight and microgravity response of the Arabidopsis plant transcriptome." npj Microgravity. 2023 Mar 20;9(1):21. http://dx.doi.org/10.1038/s41526-023-00247-6 ; PubMed PMID: 36941263; PubMed Central PMCID: PMC10027818 , Mar-2023
Project Title:  Dissecting Beneficial Plant-Microbe Interactions and Their Efficacy in the ISS Spaceflight Environment, a Model Study Reduce
Images: icon  Fiscal Year: FY 2023 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Microbiology   | Plant Biology  
Start Date: 01/01/2020  
End Date: 12/31/2023  
Task Last Updated: 03/02/2023 
Download report in PDF pdf
Principal Investigator/Affiliation:   Lewis, Norman G Ph.D. / Washington State University 
Address:  Institute of Biological Chemistry 
299 Clark Hall 
Pullman , WA 99164-6340 
Email: lewisn@wsu.edu 
Phone: 509-335-2682  
Congressional District:
Web: http://ibc.wsu.edu/research-faculty/lewis/  
Organization Type: UNIVERSITY 
Organization Name: Washington State University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Davin, Laurence  Ph.D. Washington State University, Pullman 
Kahn, Michael  Ph.D. Washington State University, Pullman 
Project Information: Grant/Contract No. 80NSSC19K1484 
Responsible Center: NASA KSC 
Grant Monitor: Levine, Howard  
Center Contact: 321-861-3502 
howard.g.levine@nasa.gov 
Unique ID: 12525 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1484 
Project Type: FLIGHT 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
(3) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: End date changed to 12/31/2023 per NSSC information (Ed, 1/15/23)

Task Description: Significance of objectives to NASA and this Solicitation: Deep space exploration or extraterrestrial colonization (e.g., Moon or Mars) will require the ability to sustainably produce plants for human/animal consumption, as well as providing aesthetic benefits of plant life to future crews and personnel in extra-terrestrial colonies. One key challenge in spaceflight/microgravity is in overcoming long-standing difficulties in efficaciously providing water and nutrients to germinating and maturing plants. Another important research challenge that has seen little attention is in productively exploiting beneficial plant-microbe interactions in spaceflight/microgravity, particularly for nitrogen (N) fixation. When both challenges are resolved for optimal, productive, and efficacious plant growth in space, this will provide the exciting opportunity to recycle organically bound carbon (C) and N that was sequestered in these plants. Through subsequent recycling of those organics (e.g., derived from human and animal consumption waste and from unused plant parts), this will help enable sustainable plant growth over multiple generations. Another benefit of studying beneficial plant microbe interactions is at the fundamental science level, i.e., by gaining much improved understanding of how the spaceflight/microgravity environment affects this important physiological process.

Central objectives of proposed research: Complementary purposes of our 2 Specific Aims are to initially dissect, understand, and optimize plant growth/development in spaceflight/microgravity via exploiting beneficial plant-microbe interactions. Then to ultimately recycle organic C and N from them suitable for subsequent multiple plant generations. To do this, we will use model Medicago plant species (e.g., alfalfa), and its beneficial bacterial symbiont, which together can potentially displace the need for N-containing fertilizer in spaceflight/microgravity.

Specific Aims:

1. Comprehensively compare and contrast efficacy of beneficial symbiotic plant-microbe interactions between Medicago and Sinorhizobium growing on the International Space Station (ISS) and on Earth (NASA Kennedy Space Center/KSC), including understanding changes occurring at the molecular level;

2. Compare and contrast ease of biodegradation of the ISS and Earth grown Medicago plant material, in order to assess whether there are any differences in the ability to recycle C, N, etc. for multiple generations of plant growth/development.

Justification for this work is threefold: The first is to demonstrate that beneficial plant microbe interactions during N-fixation can be efficaciously achieved in spaceflight/microgravity. The second is to gain a fundamental understanding of spaceflight/microgravity environment effects on these beneficial plant microbe interactions, and their potential usage for deep space exploration and colonization. The third is to demonstrate that organic C and N can be facilely recycled to support multiple generations of plant growth in space.

Methods/techniques: During growth, FluorPen and plant size measurements will be carried out to assess comparative N-fixation efficacy for each condition, both on the ISS and on Earth. Tissues (leaf, stem, and root) from the ISS and Earth control will be collected after ca. 6-8 weeks growth, frozen (-160°C). They will be subjected to transcriptomic and metabolomic (including amino acid) analyses; the microbiomes present in aerial/underground tissues will be determined. The multi-omics approaches employed are as for our Arabidopsis study.

Medicago plant material, from the ISS and ground control, will also be subjected to biodegradation to establish whether there are any differences in N-mineralization (for recycling) in spaceflight/microgravity or ground control tissues.

Research Impact/Earth Benefits: Among the benefits on Earth envisaged: improving our knowledge of N-fixing process and the symbiosis between Medicago and Sinorhizobium, and determining optimal lignin contents for space and Earth will be very instructive, as will the recycling C/N capabilities for both wild type and genetically modified plant lines. Demonstrating this in space is also a very effective means of demonstrating to aspiring young scientists (including Middle and High School students) and others of the importance of plant life, of N-fixation, and of C/N recycling in a sustainable manner.

Task Progress & Bibliography Information FY2023 
Task Progress: 1.1. Alfalfa-Sinorhizobium symbiosis in Passive Orbital Nutrient Delivery System (PONDS) and APEX (Advanced Plant Experiment) Systems – initial experiments

As described in the previous progress report, growth of alfalfa (M. sativa), together with its symbiont S. meliloti, was quite extensively evaluated in both PONDS and APEX systems, using the alfalfa Ladak cultivar and S. meliloti, as well as with commercial low lignin and normal (wild type, WT) lignin level alfalfa lines. In our ground-based studies extending until early Spring 2022, conditions were identified for satisfactory symbiotic growth/development of alfalfa with S. meliloti using the PONDS system, and we were thus poised to advance to the Science Verification Test (SVT) phase.

As an example of our progress, since carbon dioxide (CO2) levels on the International Space Station (ISS) average 3,500 ppm with relative humidity (RH) levels consistently at 45-50%, we next evaluated alfalfa growth conditions in combination with previously optimized lighting levels determined at “normal” CO2 and lower ambient humidity. Alfalfa plants were grown in magenta containers. After 6 weeks, alfalfa plants grown under 3,500 ppm CO2/45% RH conditions grew significantly taller, with larger diameter and denser stems than those of plants grown in a 410 ppm CO2 atmosphere and 30% RH. These results indicated that the environment on board the ISS was suitable for growing alfalfa, at least with respect to RH and CO2 levels.

However, our work using PONDS had to be abruptly abandoned as an unrelated (to us) PONDS test study on the ISS failed in early 2022; this information being provided by NASA Kennedy Space Center (KSC) research personnel that were associated with the PONDS ISS test. We were also informed by NASA personnel that the PONDS system would now no longer be available for our proposed ISS study. (More recently, while a modified PONDS system re-flight has been rescheduled for further ISS hardware testing in 2023, we were advised by NASA to develop other systems for our proposed ISS study.)

1.2. Re-focus on APEX plant growth chambers for ISS

This unexpectedly brought us back to further development of the APEX system to meet our ISS study goals which had previously been sub-optimal using the APEX equipment. The timeframe from Spring 2022 to December 2022 was thus focused upon establishing conditions that would allow for efficient symbiosis and alfalfa-root nodule formation, as well as for satisfactory growth of low and normal lignin level lines, in the APEX system. (An alternate configuration of APEX was thus designed.) Experiments with this APEX configuration resulted in somewhat better growth of alfalfa containing a developed root system with N-fixing nodules, although the plants did not grow to the same levels obtained when N was added as a supplement.

Since then, ground testing of alfalfa growth conditions in APEX hardware has culminated in near full completion of our final definition experiment leading up to SVT (i.e., which focuses both on satisfactory symbiosis as well as in generating sufficient alfalfa plant tissue for metabolomics, transcriptomics, lignin and lignin biodegradation analyses, etc.). To do this, twenty-four APEX Growth Chambers (AGCs) were assembled. In one experiment, 6 AGCs contained low-lignin alfalfa seed, and 6 AGCs contained a WT alfalfa line with WT lignin levels. All AGCs in that experiment had an added nitrogen source. The 12 AGCs containing these seeds were placed in a surrogate VEGGIE growth chamber, with the experiment initiated by adding a nutrient solution followed by water as needed for the duration of the plant growth. After 37 days of growth, both alfalfa lines grew as expected. In the S. meliloti-alfalfa symbiosis experiment, the other 12 AGCs contained WT alfalfa cv. Ladak seed. To test the effectiveness of the symbiosis, 6 AGCs contained S. meliloti inoculum, 4 AGCs contained an added the nitrogen source (positive controls), and 2 AGCs (negative controls) did not contain any nitrogen or S. meliloti. The 12 AGCs were also placed in a surrogate VEGGIE growth chamber. Again, plant growth was initiated by adding a nutrient solution followed by water as needed for duration of the experiment. After 37 days of growth, alfalfa inoculated with S. meliloti showed reasonably good plant growth, i.e., as expected in an effective symbiosis, whereas the controls showed either good growth (positive controls) or poor growth (negative controls).

1.3. Lignin-reduced alfalfa lines

As described previously, application of CRISPR/Cas9 was carried out to generate lignin-reduced alfalfa lines, as well as growing the same low-lignin and normal alfalfa seed described above. All lines produced were grown to assess their relative growth/development and lignin-reduction characteristics.

1.3.1. Genetic engineering for lignin-reduced alfalfa

Our previous studies involving downregulation of arogenate dehydratase (ADT) genes in Arabidopsis thaliana resulted in plants with reduced lignin levels. Initially, as described earlier, we obtained ADT homologs from M. truncatula followed by corresponding genes from cDNA preparations of M. sativa variety “Ladak” 4-week-old leaf tissue total RNA.

The CRISPR/Cas9 gene-editing approach was next selected to disable ADT genes, potentially allowing for multiple gene knock-out targets in a single experiment. Currently, we are screening regenerated transgenic plants to establish mutations in targeted genes. Transgenic CRISPR/Cas9 plants that showed promising results had sequence-confirmed CRISPR/Cas9 targeted T-DNA inserts integrated into their genomic DNA. Since alfalfa is a tetraploid having potentially four allelic variations for a given gene, specific in-depth sequencing analysis is both needed and being conducted to determine the extent of CRISPR/Cas9-targeted gene DNA modification that has occurred in each allele for the ADT gene targeted. Sequencing of polymerase chain reaction (PCR) amplicons indicated mutations in targeted sites of one or all four alleles for the specific gene. Transgenic CRISPR/Cas9 alfalfa plants were bagged for self-pollination to fix the desirable traits. Seeds from transgenic T0 plants were plated onto selective medium to obtain T1 generation plants with fixed traits of interest for further analysis and determination of extent of inheritance of targeted gene traits. T1 plants are currently growing in the greenhouse and stems have been harvested for additional lignin analysis. Flowers are being bagged for the next round of self-pollination to enhance the fixation of the desired traits in the T2 generation.

1.3.2. Lignin analyses of alfalfa plants

Lignin estimates in APEX system: Lignin estimates of individual alfalfa plant lines were performed. Estimated lignin levels of the low lignin alfalfa plants were lower as compared to the normal (WT) lignin lines grown.

Lignin estimates of CRISPR/Cas9 transgenic alfalfa: Lignin estimate analyses were preliminarily performed using WT and transgenic alfalfa greenhouse grown. Estimated lignin levels of transgenic alfalfa plants were slightly lower than WT lines.

2. Future Work

The next phase of our research is to sequentially complete the SVT and Experimental Verification Test experiments at NASA Kennedy Space Center (KSC), in preparation for approval and completion of our proposed ISS investigation.

3. Bibliography

a. Corea, O.R., Ki, C., Cardenas, C.L., Kim, S.J., Brewer, S.E., Patten, A.M., Davin, L.B., and Lewis, N.G. (2012) Arogenate dehydratase isoenzymes profoundly and differentially modulate carbon flux into lignins. J. Biol. Chem. 287: 11446-11459. http://dx.doi.org/10.1074/jbc.M111.322164

b. Hixson, K.K., Marques, J.V., Wendler, J.P., McDermott, J.E., Weitz, K.K., Clauss, T.R., Monroe, M.E., Moore, R.J., Brown, J., Lipton, M.S., Bell, C.J., Pasa Tolic, L., Davin, L.B., and Lewis, N.G. (2021) New insights into lignification via network and multi-omics analyses of arogenate dehydratase knock-out mutants in Arabidopsis thaliana. Front. Plant Sci. 12: 664250. http://dx.doi.org/10.3389/fpls.2021.664250

Bibliography: Description: (Last Updated: 11/28/2023) 

Show Cumulative Bibliography
 
Abstracts for Journals and Proceedings Mortimer MW, Lewis NG, Kahn ML, Davin, LB. "An alternative moisture and nutrient delivery method for the PONDS plant growth apparatus." 38th Annual Meeting of the American Society for Gravitational and Space Research, Houston, TX, November 9-12, 2022.

Abstracts. 38th Annual Meeting of the American Society for Gravitational and Space Research, Houston, TX, November 9-12, 2022. , Nov-2022

Abstracts for Journals and Proceedings Heinse R, Monje O, Richards J, Dufour N, Lewis, NG "Root-zone water stress assessment for the Advanced Plant Habitat (APH) inaugural mission on the International Space Station (ISS)" Committee on Space Research (COSPAR) 2022, 44th Scientific Assembly, Athens, Greece, July 16-24, 2022.

Abstracts. Committee on Space Research (COSPAR) 2022, 44th Scientific Assembly, Athens, Greece, July 16-24, 2022. , Jul-2022

Abstracts for Journals and Proceedings Lewis NG "Integrated multi-omics analyses of lignin-reduced Arabidopsis lines on International Space Station." Phytochemical Society of North America (PSNA) 2022, 61st Annual Meeting, Blacksburg, VA, July 24-28, 2022.

Abstracts. Phytochemical Society of North America (PSNA) 2022, 61st Annual Meeting, Blacksburg, VA, July 24-28, 2022. , Jul-2022

Abstracts for Journals and Proceedings Lewis NG, Davin LB, Costa MA, Moinuddin SGA, Hanschen ER, Starkenburg SR, Koehler SI, Winnacott BJ, Hixson KK, Lipton MS, Hanson DT, Turpin MM, Monje O, Richards JT, Dufour N, Levine HG. "Integrated multi-omics analyses of lignin-reduced Arabidopsis lines on International Space Station." 38th Annual Meeting of the American Society for Gravitational and Space Research, Houston, TX, November 9-12, 2022.

Abstracts. 38th Annual Meeting of the American Society for Gravitational and Space Research, Houston, TX, November 9-12, 2022. , Nov-2022

Articles in Peer-reviewed Journals Overbey EG, Saravia-Butler AM, Zhang Z, Rathi KS, Fogle H, da Silveira WA, Barker RJ, Bass JJ, Beheshti A, Berrios DC, Blaber EA, Cekanaviciute E, Costa HA, Davin LB, Fisch KM, Gebre SG, Geniza M, Gilbert R, Gilroy S, Hardiman G, Herranz R, Kidane YH, Kruse CPS, Lee MD, Liefeld T, Lewis NG, McDonald JT, Meller R, Mishra T, Perera IY, Ray S, Reinsch SS, Rosenthal SB, Strong M, Szewczyk NJ, Tahimic CGT, Taylor DM, Vandenbrink JP, Villacampa A, Weging S, Wolverton C, Wyatt SE, Zea L, Costes SV, Galazka JM. "NASA GeneLab RNA-Seq Consensus Pipeline: Standardized processing of short-read RNA-Seq data." iScience. 2021 Mar 26;24:102361. http://dx.doi.org/10.1016/j.isci.2021.102361 , Mar-2022
Articles in Peer-reviewed Journals Barker RJ, Kruse CPS, Johnson C, Saravia-Butler A, Fogle H, Chang H-s, Møller Trane R, Kinscherf N, Villacampa A, Manzano A, Herranz R, Davin LB, Lewis NG, Perera I, Wolverton C, Gupta P, Jaiswal P, Reinsch SS, Wyatt S, Gilroy S. "Meta-analysis of the space flight and microgravity response of the Arabidopsis plant transcriptome." npj Microgravity. 2023 Mar 20;9(21). http://dx.doi.org/10.1038/s41526-023-00247-6 , Mar-2023
Project Title:  Dissecting Beneficial Plant-Microbe Interactions and Their Efficacy in the ISS Spaceflight Environment, a Model Study Reduce
Images: icon  Fiscal Year: FY 2022 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Microbiology   | Plant Biology  
Start Date: 01/01/2020  
End Date: 12/31/2022  
Task Last Updated: 01/11/2022 
Download report in PDF pdf
Principal Investigator/Affiliation:   Lewis, Norman G Ph.D. / Washington State University 
Address:  Institute of Biological Chemistry 
299 Clark Hall 
Pullman , WA 99164-6340 
Email: lewisn@wsu.edu 
Phone: 509-335-2682  
Congressional District:
Web: http://ibc.wsu.edu/research-faculty/lewis/  
Organization Type: UNIVERSITY 
Organization Name: Washington State University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Davin, Laurence  Ph.D. Washington State University, Pullman 
Kahn, Michael  Ph.D. Washington State University, Pullman 
Project Information: Grant/Contract No. 80NSSC19K1484 
Responsible Center: NASA KSC 
Grant Monitor: Levine, Howard  
Center Contact: 321-861-3502 
howard.g.levine@nasa.gov 
Unique ID: 12525 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1484 
Project Type: FLIGHT 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
(3) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: None
Task Description: Significance of objectives to NASA and this Solicitation: Deep space exploration or extraterrestrial colonization (e.g., Moon or Mars) will require the ability to sustainably produce plants for human/animal consumption, as well as providing aesthetic benefits of plant life to future crews and personnel in extra-terrestrial colonies. One key challenge in spaceflight/microgravity is in overcoming long-standing difficulties in efficaciously providing water and nutrients to germinating and maturing plants. Another important research challenge that has seen little attention is in productively exploiting beneficial plant-microbe interactions in spaceflight/microgravity, particularly for nitrogen (N) fixation. When both challenges are resolved for optimal, productive, and efficacious plant growth in space, this will provide the exciting opportunity to recycle organically bound carbon (C) and N that was sequestered in these plants. Through subsequent recycling of those organics (e.g., derived from human and animal consumption waste and from unused plant parts), this will help enable sustainable plant growth over multiple generations. Another benefit of studying beneficial plant microbe interactions is at the fundamental science level, i.e., by gaining much improved understanding of how the spaceflight/microgravity environment affects this important physiological process.

Central objectives of proposed research: Complementary purposes of our 2 Specific Aims are to initially dissect, understand, and optimize plant growth/development in spaceflight/microgravity via exploiting beneficial plant-microbe interactions. Then to ultimately recycle organic C and N from them suitable for subsequent multiple plant generations. To do this, we will use model Medicago plant species (e.g., alfalfa), and its beneficial bacterial symbiont, which together can potentially displace the need for N-containing fertilizer in spaceflight/microgravity.

Specific Aims:

1. Comprehensively compare and contrast efficacy of beneficial symbiotic plant-microbe interactions between Medicago and Sinorhizobium growing on the International Space Station (ISS) and on Earth (NASA Kennedy Space Center/KSC), including understanding changes occurring at the molecular level;

2. Compare and contrast ease of biodegradation of the ISS and Earth grown Medicago plant material, in order to assess whether there are any differences in the ability to recycle C, N, etc. for multiple generations of plant growth/development.

Justification for this work is threefold: The first is to demonstrate that beneficial plant microbe interactions during N-fixation can be efficaciously achieved in spaceflight/microgravity. The second is to gain a fundamental understanding of spaceflight/microgravity environment effects on these beneficial plant microbe interactions, and their potential usage for deep space exploration and colonization. The third is to demonstrate that organic C and N can be facilely recycled to support multiple generations of plant growth in space.

Methods/techniques: During growth, FluorPen and plant size measurements will be carried out to assess comparative N-fixation efficacy for each condition, both on the ISS and on Earth. Tissues (leaf, stem, and root) from the ISS and Earth control will be collected after ca. 6-8 weeks growth, frozen (-160°C). They will be subjected to transcriptomic, proteomic, and metabolomic (including amino acid) analyses; the microbiomes present in aerial/underground tissues will be determined. The multi-omics approaches employed are as for our Arabidopsis study.

Medicago plant material, from the ISS and ground control, will also be subjected to biodegradation to establish whether there are any differences in N-mineralization (for recycling) in spaceflight/microgravity or ground control tissues.

Research Impact/Earth Benefits: Among the benefits on Earth envisaged: improving our knowledge of N-fixing process and the symbiosis between Medicago and Sinorhizobium, and determining optimal lignin contents for space and Earth will be very instructive, as will the recycling C/N capabilities for both wild type and genetically modified plant lines. Demonstrating this in space is also a very effective means of demonstrating to aspiring young scientists (including Middle and High School students) and others of the importance of plant life, of N-fixation, and of C/N recycling in a sustainable manner.

Task Progress & Bibliography Information FY2022 
Task Progress: 1. Evaluation of Passive Orbital Nutrient Delivery System (PONDS) and APEX (Advanced Plant Experiment) Systems

1.1. Establishing N-fixing symbiosis on the ISS

Responsibilities here included trying to anticipate and solve problems that might arise in translating ground-based procedures used to set up N-fixing symbiosis with Medicago species to formats used on the ISS. We have made considerable progress and are nearing the point where plausible procedures are contemplated for the ISS. Major issues faced were choice and validation of plants and symbionts, use of NASA certified growth platforms, and establishing routine methods for plant growth and periodic monitoring.

1.2. Choice of plants and symbionts

We originally identified Medicago sativa and M. truncatula as candidate plant species, together with their respective bacterial symbionts, Sinorhizobium meliloti and S. medicae. M. truncatula is a diploid model for alfalfa, a major forage crop, and there were significant reasons for its consideration. However, M. truncatula grew poorly in containers and was more sensitive to irregularities in light. We therefore chose alfalfa, since it is generally more robust, and established appropriate growing conditions with its Ladak cultivar, which is routinely grown in the western part of the United States. Ladak forms a good symbiosis with S. medicae Rm1021, and there is considerable literature describing the bacterial symbiosis with various alfalfa cultivars. We also have recently shown that Rm1021 forms a satisfactory symbiosis with a low-lignin alfalfa strain, and a related one with normal levels of alfalfa lignin.

1.3. Optimizing plant growth hardware and conditions for conducting an effective alfalfa-Sinorhizobium symbiosis study on ISS

LED lighting and experimental conditions: We established a small facility for testing various LED lighting conditions to determine optimal light intensity and color ratio for alfalfa growth. As carbon dioxide (CO2) levels on the ISS average 3,500 ppm with relative humidity (RH) levels consistently at 45-50%, we tested both conditions in combination with previously optimized lighting levels determined at “normal” CO2 and lower ambient humidity to evaluate alfalfa growth. After 6 weeks, alfalfa plants grown in 3500 ppm CO2/45% RH grew significantly taller, with larger diameter, denser stems than those of plants grown in a 410 ppm CO2 and 30% RH. These results indicate that the environment on board the ISS is suitable for growing alfalfa, at least with respect to humidity and CO2.

APEX plant growth chambers: NASA-supplied APEX growth containers were evaluated for the ability to grow alfalfa in symbiosis with S. meliloti. Experiments with the APEX configuration gave good growth of alfalfa, containing a developed root system with N-fixing nodules.

PONDS plant growth hardware: NASA-supplied PONDS plant growth containers were tested for the ability to grow alfalfa in symbiosis with S. meliloti Rm 1021 under previously optimized growing conditions. PONDS plant containers produced healthy plants after 6 weeks of growth, and contained N-fixing nodules of normal size, shape, and color.

Inoculation techniques: In typical ground-based symbiosis experiments, two-day old seedlings were hand inoculated with Rhizobium symbiont. However, ISS experiments require that inoculum be included in pre-packaged plant growth hardware. We tested several different inoculation strategies, all of which were successful. These included: lyophilized Rhizobium powder applied to seeds or wicks; Rhizobium infused guar gum glue; seeds coated with Rhizobium paste; and wicks soaked in bacterial cultures then dried. More experiments will determine which technique will be selected for the ISS experiment, but the current data indicate a very high probability of succeeding.

2. Lignin-Reduced Alfalfa

We have taken 2 approaches to obtain lignin-reduced alfalfa lines. The first lignin-reduced alfalfa line was obtained as seed, and the second deploys CRISPR/Cas9 to generate lignin-reduced alfalfa.

2.1. Low lignin alfalfa

Low lignin alfalfa is being grown from seed to assess their growth/development and lignin-reduction characteristics in the configurations above, with comparison to another alfalfa line of ‘normal’ lignin content.

2.2. Genetic engineering for lignin-reduced alfalfa

2.2.1. Cloning

Previous studies in our lab involving downregulation of arogenate dehydratase (ADT) genes in Arabidopsis thaliana resulted in reduced lignin plants (1,2). [Ed. Note: footnotes correspond to References at the end of the Task Progress section.] Therefore, we searched the National Center for Biotechnology Information (NCBI) database for homologs to these ADT genes in Medicago, since the latter was to be used for our currently planned ISS experiments. Initially, we obtained ADT homologs from Medicago truncatula and then later isolated the corresponding genes from cDNA preparations of M. sativa.

M. sativa variety “Ladak” plants were grown in sterile tissue culture containers in Murashige and Skoog (MS) agar medium. Total RNA and then DNA were prepared from 4-week-old leaves, this cDNA being used in RT-PCR experiments to isolate ADT homologs with primers designed from M. truncatula ADT.

CRISPR/Cas9 gene-editing was selected to disable ADT genes, potentially allowing for multiple gene knock-out targets in a single effort. Sequences were submitted to the Medicago datasets in CRISPRdirect and other plant CRISPR databases to detect and select gRNAs to target specific sequences for CRISPR/Cas9 genome editing with minimal off-targeting.

Methods developed by the Voytas lab (University of Minnesota) were used to clone multiple targets into a single T-DNA binary vector containing Csy4 binding sites and gRNA repeat regions, driven by the CmYLCV promoter (3). [Ed. Note: see References at end of Task Progress section.] The vector also contains the Cas9 enzyme driven by the 35S promoter, and final assembled vector constructs were sequenced to confirm that all components of the reaction were present. Confirmed vectors were transformed into Agrobacterium tumefaciens and verified by additional polymerase chain reaction (PCR) screening and resequencing.

Callus formation/plant regeneration/selection: Ladak cultivar plants were germinated and maintained in sterile conditions in Magenta box containers to provide leaf material for co-cultivation with Agrobacterium. Co-cultivated plant leaves were placed on medium containing hormones to induce callus development, with ticarcillin antibiotic to eliminate Agrobacterium after co-cultivation; neomycin phosphotransferase (npt) plant selectable marker was used for kanamycin resistance. Generated calli were then transferred to medium for shoot initiation, followed by plantlet formation. Plantlets were transferred to rooting medium and transferred to the greenhouse after rooting was sufficiently developed.

Currently, we are screening regenerated transgenic plants to detect mutations in targeted genes. Plants are being checked by PCR analysis to confirm presence of the npt gene selectable marker, the T-DNA insert, and specific ADT3 or ADT6 genes to use for subcloning for sequence analysis.

2.2.2. Transgenic plant screening approaches to detect CRISPR/Cas9 mutations

The tetraploid nature of alfalfa requires additional methods to confirm mutated targeted regions in the ADT genes. This includes restriction digestion of PCR amplicons, and primer sets for annealing to targeted regions to detect mutations.

Transgenic plant PCR amplified products are currently being cloned into a pCR4-TOPO vector to screen individual colonies for sequencing to detect variations in all four alleles of the tetraploid alfalfa. These results will be used to confirm allelic mutations arising in the ADT genes targeted in transgenic plants.

2.3. Metabolomic and lignin analyses of alfalfa lines

Metabolomic analyses for different tissues of wild type (WT) alfalfa plant lines, grown under two different light regimens, were carried out. Initially, ~3 to 4 plants of 5 week old alfalfa plants grown under 18- and 24-hour light schedules were selected. Alfalfa plants grown under the 24-hour lighting had ~1.1 × higher plant biomass compared to those grown under 18-hour lighting.

Metabolite extraction and analyses of leaf, stem, root, and nodule tissues from each plant were performed. The aqueous methanol extracts of individual tissue samples, obtained after initially pulverizing them with liquid nitrogen and later sonicating with MeOH: H2O, were subjected to UPLC-qTOF-MS for metabolite analyses.

Following liquid chromatography–mass spectrometry (LC-MS) analysis, identification of metabolites in each alfalfa plant was performed after normalization of metadata to the internal standard naringenin, followed by comparing their masses with reported literature data. The major metabolites (putative identification) were flavones in leaves (~44), stems (~35), roots (~30), and nodules (~25), respectively.

To determine if there was any significant variation in relative metabolite levels identified in each alfalfa tissue sample grown under 18- and 24-hour lighting, a statistical analysis -- i.e., orthogonal partial least squares discriminant analysis (OPLSDA) and heatmaps -- using web server MetaboAnalyst 5.0 was performed. OPLSDA plots of each individual tissue sample showed a clear segregation of metabolite clusters for plants grown under 18- and 24-hour light regimens under the growth conditions employed. Next, lignin estimates were performed to determine their levels in alfalfa plants grown under 18- and 24-hour lighting regimens. Pulverized individual stem samples were subjected to organic solvent extraction, followed by a water wash, then freeze-dried and later subjected to thioacidolysis by treating with dioxane-ethanethiol in presence of boron trifluoride etherate at 100 °C. Monomeric thioacidolysis derived products released from each sample were quantified, with estimated lignin levels of the alfalfa plants grown under 24-hour lighting having very slightly lower content (may not be statistically significant) as compared to both 18-hour lighting and greenhouse growth conditions.

References

1. Corea, O.R., Ki, C., Cardenas, C.L., Kim, S.J., Brewer, S.E., Patten, A.M., Davin, L.B., and Lewis, N.G. (2012) Arogenate dehydratase isoenzymes profoundly and differentially modulate carbon flux into lignins. J. Biol. Chem. 287: 11446-11459. http://dx.doi.org/10.1074/jbc.M111.322164

2. Hixson, K.K., Marques, J.V., Wendler, J.P., McDermott, J.E., Weitz, K.K., Clauss, T.R., Monroe, M.E., Moore, R.J., Brown, J., Lipton, M.S., Bell, C.J., Pasa Tolic, L., Davin, L.B., and Lewis, N.G. (2021) New insights into lignification via network and multi-omics analyses of arogenate dehydratase knock-out mutants in Arabidopsis thaliana. Front. Plant Sci. 12: 664250. http://dx.doi.org/10.3389/fpls.2021.664250

3. Cermak, T., Curtin, S.J., Gil-Humanes, J., Cegan, R., Kono, T.J.Y., Konecna, E., Belanto, J.J., Starker, C.G., Mathre, J.W., Greenstein, R.L., and Voytas, D.F. (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29: 1196-1217. http://dx.doi.org/10.1105/tpc.16.00922

Bibliography: Description: (Last Updated: 11/28/2023) 

Show Cumulative Bibliography
 
 None in FY 2022
Project Title:  Dissecting Beneficial Plant-Microbe Interactions and Their Efficacy in the ISS Spaceflight Environment, a Model Study Reduce
Images: icon  Fiscal Year: FY 2021 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Microbiology   | Plant Biology  
Start Date: 01/01/2020  
End Date: 12/31/2022  
Task Last Updated: 03/02/2021 
Download report in PDF pdf
Principal Investigator/Affiliation:   Lewis, Norman G Ph.D. / Washington State University 
Address:  Institute of Biological Chemistry 
299 Clark Hall 
Pullman , WA 99164-6340 
Email: lewisn@wsu.edu 
Phone: 509-335-2682  
Congressional District:
Web: http://ibc.wsu.edu/research-faculty/lewis/  
Organization Type: UNIVERSITY 
Organization Name: Washington State University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Davin, Laurence  Ph.D. Washington State University, Pullman 
Kahn, Michael  Ph.D. Washington State University, Pullman 
Project Information: Grant/Contract No. 80NSSC19K1484 
Responsible Center: NASA KSC 
Grant Monitor: Levine, Howard  
Center Contact: 321-861-3502 
howard.g.levine@nasa.gov 
Unique ID: 12525 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1484 
Project Type: FLIGHT 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
(3) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: None
Task Description: Significance of objectives to NASA and this Solicitation: Deep space exploration or extraterrestrial colonization (e.g., Moon or Mars) will require the ability to sustainably produce plants for human/animal consumption, as well as providing aesthetic benefits of plant life to future crews and personnel in extra-terrestrial colonies. One key challenge in spaceflight/microgravity is in overcoming long-standing difficulties in efficaciously providing water and nutrients to germinating and maturing plants. Another important research challenge that has seen little attention is in productively exploiting beneficial plant-microbe interactions in spaceflight/microgravity, particularly for nitrogen (N) fixation. When both challenges are resolved for optimal, productive, and efficacious plant growth in space, this will provide the exciting opportunity to recycle organically bound carbon (C) and N that was sequestered in these plants. Through subsequent recycling of those organics (e.g., derived from human and animal consumption waste and from unused plant parts), this will help enable sustainable plant growth over multiple generations. Another benefit of studying beneficial plant microbe interactions is at the fundamental science level, i.e., by gaining much improved understanding of how the spaceflight/microgravity environment affects this important physiological process.

Central objectives of proposed research: Complementary purposes of our 2 Specific Aims are to initially dissect, understand, and optimize plant growth/development in spaceflight/microgravity via exploiting beneficial plant-microbe interactions. Then to ultimately recycle organic C and N from them suitable for subsequent multiple plant generations. To do this, we will use model Medicago plant species (e.g., alfalfa), and its beneficial bacterial symbiont, which together can potentially displace the need for N-containing fertilizer in spaceflight/microgravity.

Specific Aims:

1. Comprehensively compare and contrast efficacy of beneficial symbiotic plant-microbe interactions between Medicago and Sinorhizobium growing on International Space Station (ISS) and on Earth (NASA Kennedy Space Center/KSC), including understanding changes occurring at the molecular level;

2. Compare and contrast ease of biodegradation of ISS and Earth grown Medicago plant material, in order to assess whether there are any differences in the ability to recycle C, N, etc. for multiple generations of plant growth/development.

Justification for this work is threefold: The first is to demonstrate that beneficial plant microbe interactions during N-fixation can be efficaciously achieved in spaceflight/microgravity. The second is to gain a fundamental understanding of spaceflight/microgravity environment effects on these beneficial plant microbe interactions, and their potential usage for deep space exploration and colonization. The third is to demonstrate that organic C and N can be facilely recycled to support multiple generations of plant growth in Space.

Methods/techniques: During growth, FluorPen and plant size measurements will be carried out to assess comparative N-fixation efficacy for each condition, both on ISS and Earth. Tissues (leaf, stem, and root) from ISS and Earth control will be collected after ca. 6-8 weeks growth, frozen (-160°C). They will be subjected to transcriptomic, proteomic, and metabolomic (including amino acid) analyses; the microbiomes present in aerial/underground tissues will be determined. The multi-omics approaches employed are as for our Arabidopsis study.

Medicago plant material, from ISS and ground control, will also be subjected to biodegradation to establish whether there are any differences in N-mineralization (for recycling) in spaceflight/microgravity or ground control tissues.

Research Impact/Earth Benefits: Among the benefits on Earth envisaged, improving our knowledge of N-fixing process and the symbiosis between Medicago and Sinorhizobium, and determining optimal lignin contents for Space and Earth will be very instructive, as will the recycling C/N capabilities for both wild type and genetically modified plant lines. Demonstrating this in Space is also a very effective means of demonstrating to aspiring young scientists (including Middle and High School students) and others of the importance of plant life, of N-fixation, and of C/N recycling in a sustainable manner.

Task Progress & Bibliography Information FY2021 
Task Progress: Two Medicago species were considered for the ISS study, namely Medicago sativa (alfalfa) and Medicago truncatula (barrel medic). Alfalfa is a common forage and is well studied. However, it is also an outcrossing tetraploid that has largely been displaced as a model legume by barrel medic. The latter is a self-fertilizing diploid.

Various experiments have been carried out to date to evaluate suitability of both species for ISS. Our focus has been on both hardware and manipulations needed for the proposed ISS study, i.e., in order to grow plants to a growth/developmental stage where we can convincingly demonstrate and evaluate successful nitrogen (N)-fixation and lignification in the microgravity environment.

For the work described below, we have productive monthly teleconferences with Kennedy Space Center personnel to discuss our ongoing experimental strategy and results obtained.

Progress made to date is as follows:

Wicks: Key to our proposed experiments on ISS is the use of wicks, which both hold the seeds of Medicago species during launch and also provide an effective means of enabling watering the plants for both germination and growth/development.

The wicks also have a crucial role for all of our downstream experiments, including for facile removal of plant shoots and roots from the plant growth containers. This is because the wicks allow for not only ready removal of root tissue, but also enable this to occur with the intact nodules in place. The wick experimental design can allow for both separation of nodules from the support medium and also for them to be collected quickly and efficiently.

Hardware: Another experimental design variable is potential hardware for ISS using Veggie. We are currently assessing three different types of plant growth hardware, all certified and provided by NASA. These are the PILLOWS, PONDS, and APEX hardware, respectively. [Ed. Note: For more information on PILLOWS, PONDS, and APEX, see links referenced below.] All three have advantages and disadvantages. Our experiments are additionally designed to identify exactly what conditions and arrangements are best suited for our experiments to best succeed on the ISS.

Using these three different forms of hardware, we have been testing and evaluating both plant species for their suitability to germinate and grow/develop in each, including establishing effects of light levels on growth/development, on nodulation, and on effects of ethylene with ethylene insensitive M. truncatula mutants. Additional work is being done on generating lignin-reduced Medicago lines, and on metabolomics analyses as described below.

Germination: Alfalfa is generally easier to germinate than M. truncatula. Here, we routinely achieved good germination rates with alfalfa under conditions that mimic the wicking arrangement that we wish to deploy. However, we have not had reasonable success in getting high germination of M. truncatula under these same conditions. A second limitation we had was the APEX hardware system. While good germination occurs, the plants did not grow further. This appeared to be due to some incompatibility between Medicago and the Oasis foam material, and unrelated to the symbiosis.

Light levels: We know from previous experience that alfalfa tolerates a variety of light conditions (it can be grown in continuous light), whereas M. truncatula is more sensitive to day length. We have tested both plant species under several LED (light emitting diode) lighting conditions, and identified conditions where both species grow well. By experimenting with both LED light intensity and color balance, we identified potentially optimal light conditions for growing our plants under the constraints of the VEGGIE plant growth hardware.

Nodulation: This provided excellent results. Both Medicago species nodulated well, under some of the growth conditions tested, including in various ISS hardware configurations.

Magenta boxes and ethylene: Our team has routinely grown alfalfa in closed magenta boxes, whereas M. truncatula does not grow well. We investigated whether this difference might be due to the known greater sensitivity of M. truncatula to ethylene, a plant hormone, by testing the growth of an ethylene insensitive mutant of M. truncatula in closed boxes. The mutant M. truncatula still grew very poorly in closed boxes, indicating that it is not suitable for these conditions. We are uncertain what the explanation is but do not think alfalfa presents a similar challenge.

Lignin reduced alfalfa: An additional aspect of our experimental design is to use low lignin Medicago lines for our study on ISS. This work is progressing smoothly. The purpose here is in developing capabilities to be able to more readily biodegrade and recycle non-usable plant material, i.e., tissues which could not be used for consumption (in this case, tissues other than the usable part for forage). To do this, we need lignin-reduced lines that are more suitable for future long-duration spaceflight missions and/or for extraterrestrial colonization, i.e., where C/N recycling is optimal. Our overall strategy is to evaluate the level of C and N recovery attainable from such modified tissues via biodegradation, versus wild type lines.

We have a dual-pronged approach to obtain lignin-reduced plants. The first is to down-regulate arogenate dehydratases as previously carried out with Arabidopsis thaliana. Here we are employing CRISPR/Cas9 genome editing, as this technology allows for creation of multiple mutants in a single step, thus time-consuming crosses and/or backcrosses are not needed. We are en route to generate single and multiple gene knockouts in alfalfa by targeting a single ADT gene with two or four guide RNAs, and/or by targeting two ADT genes with specific guide RNAs. For each gene (MsADT4 and MsADT5), sgRNA was designed (Synthego CRISPR Design Tool) to mitigate the potential for low-off target potential.

M. sativa variety “LADAK” plants have been grown in sterile tissue culture containers in MS agar medium under 16 h of 120 mm fluorescent light conditions at 22°C. Total RNA was then prepared from 4-week-old leaves from these plants using a Sigma Spectrum Plant Total RNA Kit protocol. An Invitrogen SuperScript III First Strand Synthesis System was next used to prepare cDNA from 2 µg of the total RNA. The cDNA is currently being used in RT-PCR experiments to isolate the arogenate dehydratase gene homologs using primers designed from M. truncatula homologs that were previously identified using NCBI BLAST procedures. Once these M. sativa homologs are identified and sequenced completely, they will be subjected to analysis using the CRISPR Design Tool to identify 20 bp target DNA regions within exons for the purpose of knocking out the specific gene-of-interest. These sgRNA target regions will next be cloned into a binary vector containing the Cas9 protein and transformed into Agrobacterium tumefaciens for use in obtaining transgenic CRISPR/Cas9 lignin-reduced Medicago plants.

Metabolomic analyses of M. truncatula and alfalfa species: Initially, two 5-6 week old plants each of M. truncatula and alfalfa plant lines were individually harvested. For metabolite extraction and analyses, each individual plant (containing leaves, stems, and roots) were collected, flash frozen in liquid nitrogen, and kept at -80°C. Next, each tissue type of M. truncatula and alfalfa plant lines was individually ground to a fine powder with liquid nitrogen using a mortar and pestle. A known amount (150-200 mg × 3) of each frozen pulverized tissue sample was weighed into a 2 ml Eppendorf tube and flash frozen in liquid nitrogen, to which ice-cold MeOH:H2O (1:1) containing 0.1 M naringenin as an internal standard was individually added. Samples were then individually sonicated for 10 minutes in ice-cold water, vortexed for few seconds, and centrifuged for 1 min at 4°C (16,000g). To the contents, ice-cold chloroform was next added, sonicated for 10 minutes in ice-cold water, vortexed for few seconds, and centrifuged for 15 min at 4°C (16,000g). Each aqueous methanol extract was individually separated and subjected to UPLC-qTOF-MS for metabolite analyses using Waters Acquity Ultra Performance LC system (Waters) equipped with a photodiode array (PDA) detector (Waters) coupled to a XevoTM G2 QTof mass spectrometer (Waters MS Technologies, Manchester, UK). Each chloroform extract and pellet were then separated and stored at –80°C for further analysis. From the LC-MS analysis, identification of metabolites in M. truncatula and alfalfa plant tissue types were performed by comparing their masses with reported literature data. Some ~100 metabolites (with putative identification, mainly belonging to flavonoid and saponin natural product classes), were individually annotated from leaves, stem and roots of each Medicago species.

In sum, the alfalfa lines seem to offer the most promise for the proposed ISS studies, but we are still evaluating the hardware configuration.

Links for Further Reference

PILLOWS rooting packet: https://ntrs.nasa.gov/citations/20110015824

PONDS/Passive Orbital Nutrient Delivery System: https://www.nasa.gov/feature/the-shape-of-watering-plants-in-space

APEX/Advanced Plant Experiment: https://science.nasa.gov/biological-physical/investigations/apex-7

Bibliography: Description: (Last Updated: 11/28/2023) 

Show Cumulative Bibliography
 
 None in FY 2021
Project Title:  Dissecting Beneficial Plant-Microbe Interactions and Their Efficacy in the ISS Spaceflight Environment, a Model Study Reduce
Images: icon  Fiscal Year: FY 2020 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Microbiology   | Plant Biology  
Start Date: 01/01/2020  
End Date: 12/31/2022  
Task Last Updated: 09/11/2019 
Download report in PDF pdf
Principal Investigator/Affiliation:   Lewis, Norman G Ph.D. / Washington State University 
Address:  Institute of Biological Chemistry 
299 Clark Hall 
Pullman , WA 99164-6340 
Email: lewisn@wsu.edu 
Phone: 509-335-2682  
Congressional District:
Web: http://ibc.wsu.edu/research-faculty/lewis/  
Organization Type: UNIVERSITY 
Organization Name: Washington State University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Davin, Laurence  Ph.D. Washington State University, Pullman 
Kahn, Michael  Ph.D. Washington State University, Pullman 
Project Information: Grant/Contract No. 80NSSC19K1484 
Responsible Center: NASA KSC 
Grant Monitor: Levine, Howard  
Center Contact: 321-861-3502 
howard.g.levine@nasa.gov 
Unique ID: 12525 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1484 
Project Type: FLIGHT 
Flight Program:  
No. of Post Docs:  
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
(3) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: None
Task Description: Significance of objectives to NASA and this Solicitation: Deep space exploration or extraterrestrial colonization (e.g., Moon or Mars) will require the ability to sustainably produce plants for human/animal consumption, as well as providing aesthetic benefits of plant life to future crews and personnel in extra-terrestrial colonies. One key challenge in spaceflight/microgravity is in overcoming long-standing difficulties in efficaciously providing water and nutrients to germinating and maturing plants. Another important research challenge that has seen little attention is in productively exploiting beneficial plant-microbe interactions in spaceflight/microgravity, particularly for nitrogen (N) fixation. When both challenges are resolved for optimal, productive, and efficacious plant growth in space, this will provide the exciting opportunity to recycle organically bound carbon (C) and N that was sequestered in these plants. Through subsequent recycling of those organics (e.g., derived from human and animal consumption waste and from unused plant parts), this will help enable sustainable plant growth over multiple generations. Another benefit of studying beneficial plant microbe interactions is at the fundamental science level, i.e., by gaining much improved understanding of how the spaceflight/microgravity environment affects this important physiological process.

Central objectives of proposed research: Complementary purposes of our 2 Specific Aims are to initially dissect, understand, and optimize plant growth/development in spaceflight/microgravity via exploiting beneficial plant-microbe interactions. Then to ultimately recycle organic C and N from them suitable for subsequent multiple plant generations. To do this, we will use model Medicago plant species (e.g., alfalfa), and its beneficial bacterial symbiont, which together can potentially displace the need for N-containing fertilizer in spaceflight/microgravity.

Specific Aims:

1. Comprehensively compare and contrast efficacy of beneficial symbiotic plant-microbe interactions between Medicago and Sinorhizobium growing on International Space Station (ISS) and on Earth (NASA Kennedy Space Center/KSC), including understanding changes occurring at the molecular level;

2. Compare and contrast ease of biodegradation of ISS and Earth grown Medicago plant material, in order to assess whether there are any differences in the ability to recycle C, N, etc. for multiple generations of plant growth/development.

Justification for this work is threefold: The first is to demonstrate that beneficial plant microbe interactions during N-fixation can be efficaciously achieved in spaceflight/microgravity. The second is to gain a fundamental understanding of spaceflight/microgravity environment effects on these beneficial plant microbe interactions, and their potential usage for deep space exploration and colonization. The third is to demonstrate that organic C and N can be facilely recycled to support multiple generations of plant growth in Space.

Methods/techniques: During growth in Advanced Plant Habitat (APH), FluorPen and plant size measurements will be carried out to assess comparative N-fixation efficacy for each condition, both on ISS and Earth. Tissues (leaf, stem, and root) from ISS and Earth control will be collected after ca. 6-8 weeks growth, frozen (-160°C). They will be subjected to transcriptomic, proteomic, and metabolomic (including amino acid) analyses; the microbiomes present in aerial/underground tissues will be determined. The multi-omics approaches employed are as for our Arabidopsis study.

Medicago plant material, from ISS and ground control, will also be subjected to biodegradation to establish whether there are any differences in N-mineralization (for recycling) in spaceflight/microgravity or ground control tissues.

Research Impact/Earth Benefits:

Task Progress & Bibliography Information FY2020 
Task Progress: New project for FY2020.

Bibliography: Description: (Last Updated: 11/28/2023) 

Show Cumulative Bibliography
 
 None in FY 2020