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Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2024 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 03/15/2024  
Task Last Updated: 06/21/2024 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Su, Shih-Heng  Ph.D. University of Wisconsin-Madison 
Key Personnel Changes / Previous PI: July 2020 report: Dr. Shih-Heng Su is an Assistant Scientist in the Masson lab who has been contributing to this project since its inception.
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Flight Assignment/Project Notes: NOTE: End date changed to 03/15/2024 per the PI and per NSSC information (Ed., 6/21/24).

NOTE: End date changed to 09/15/2023 per NSSC information (Ed., 7/26/22).

Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station (ISS) and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project lead to a better understanding of the role of Putrescine and/or its derived signaling products in mitigating the stress associated with growth under microgravity environments such as those encountered during spaceflight and on the International Space Station. It also allowed the identification of novel genetic engineering strategies aimed at improving plant adaptation to the microgravity environment, with potential impact on our ability to improve the genome of other plant species / crops for utilization in bioregenerative life-support systems during spaceflight. A better understanding of plant responses to stress may also have important applications in agriculture for crop production on marginal lands.

New large-scale RNAseq expression data associated with growth under microgravity for previously untested Arabidopsis genotypes were generated during this project and will be submitted to the GeneLab Data System.

This project allowed us to introduce undergraduate students to the idiosyncrasies of scientific research in Space Biology, and also generated novel instructional materials allowing effective teaching of the difficult concepts of gravity perception by plants and their importance for agricultural production on Earth and during spaceflight. Such materials should be usable in K-12, undergraduate, and graduate education, as well as general-public outreach.

Task Progress & Bibliography Information FY2024 
Task Progress: The main objective of Advanced Plant Experiment (APEX-08) was to evaluate the impact of genetic alterations of the putrescine pool in Arabidopsis thaliana on its seedlings’ ability to grow, develop and respond to the microgravity conditions of the International Space Station (ISS) relative to ground control. To this end, we developed and characterized Arabidopsis thaliana mutant and transgenic lines with alterations in putrescine metabolism including: (1) wild type Col-0, (2) adc1-1 knockout mutant affected in its ability to synthesize putrescine; (3) Cuao3-1 and cuao3-100cdr7 allelic mutants affected in their ability to degrade putrescine; (4) transgenic plants over-expressing the Poncirus trifoliata ADC (PtADC) gene involved in putrescine synthesis (hereafter named OXPtADC), and (5) transgenic cuao3 plants over-expressing the AtCuAO3 transgene (hereafter named OXCuAO3). The PtADC over-expressing plants and Cuao3 knockout mutants were shown to accumulate more putrescine in shoot and root tissues than the other lines, allowing us to test the potential effect of increased putrescine content, or related metabolic changes, on plant responses to the stress associated with spaceflight conditions.

To evaluate the effect of alterations in the pool of putrescine and/or its catabolites on Arabidopsis thaliana seedling responses to the stress encountered during spaceflight, we germinated Arabidopsis seedlings of all six genotypes in petri dishes with the APEX growth chamber on ISS, and ran a similar control experiment on the ground, at NASA Kennedy Space Center, mimicking the growth conditions measured on ISS. At the end of this growth period, the plates were photographed for subsequent analysis of seedling growth. The seedlings were harvested, fixed in RNAlater® and returned to the laboratory for RNA extraction and expression analysis.

Results indicate that seedlings of all genotypes grown under microgravity conditions on ISS carry longer leaf petioles than those grown in a ground control at NASA Kennedy Space Center (KSC). This difference was less pronounced in putrescine over-expressing lines (OxPtADC and cuao3 mutant) than wild type. Additionally, the roots of ISS-grown seedlings were oriented more randomly than those of ground-control seedlings.

To characterize the effects of spaceflight on the patterns of gene expression, we dissected root and shoot tissues from RNAlater-fixed seedlings of all genotypes, extracted RNA from these samples, and sequenced them. The root and shoot tissues of all genotypes displayed strong gene-expression responses to the spaceflight conditions, with expression responses preferentially associated with hypoxia, reactive oxygen species (ROS) signaling, cell wall metabolism, and photosynthesis, amongst other things. Importantly, plants over-expressing the PtADC gene and cuao3 knockout plants displayed dramatically simplified transcriptional response profiles to spaceflight relative to ground control, suggesting a role for putrescine accumulation in plant tissues on their ability to tolerate the stress associated with spaceflight conditions.

Considering this important observation, we also set up to investigate the ability of plants with the same genetic alterations in the polyamine metabolic pathway to grow and develop on lunar regolith simulant (LHS-1), a substrate previously suggested to be stressful for plants. The results of preliminary seedling-viability experiments suggest plants over-expressing the PtADC transgene also display increased tolerance to lunar regolith simulants.

Taken together, our results suggest engineering plants for increased accumulation of putrescine within their tissues upon exposure to microgravity and/or lunar regoliths may confer improved tolerance to these novel environments. This observation may have important implications for the design of plant-based bioregenerative systems aimed at supporting life in long-term space exploration missions and/or exoplanet colonization programs.

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
Abstracts for Journals and Proceedings Masson PH, Su S-H, Keith M, Kinney F. "Using genome-wide association studies and RNA-seq to investigate the molecular mechanisms underlying root circumnutation in Brachypodium distachyon." PAG 31, International Plant and Animal Genome Conference, San Diego, CA, January 16, 2024.

Abstracts. PAG 31, International Plant and Animal Genome Conference, San Diego, CA, January 16, 2024. Abstract: 52206. https://pag.confex.com/pag/31/meetingapp.cgi/Paper/52206 , Jan-2024

Abstracts for Journals and Proceedings Su S-H and Masson PH "Alterations in the Polyamine Metabolic Pathway Help Alleviate the Stress Associated with Plant Exposure to Spaceflight Conditions. " Seminar

Seminar, Department of Biology, Texas State University, San Marcos, TX , Apr-2024

Awards Su S-H. "University of Wisconsin-Madison Award for Mentoring Undergraduates in Research, Scholarly and Creative Activities." May-2023
Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2023 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 09/15/2023  
Task Last Updated: 08/22/2023 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Su, Shih-Heng  Ph.D. University of Wisconsin-Madison 
Key Personnel Changes / Previous PI: July 2020 report: Dr. Shih-Heng Su is an Assistant Scientist in the Masson lab who has been contributing to this project since its inception.
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Flight Assignment/Project Notes: NOTE: End date changed to 09/15/2023 per NSSC information (Ed., 7/26/22).

Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station (ISS) and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project will lead to a better understanding of the role of Putrescine and/or its derived signaling products in mitigating the stress associated with growth under microgravity environments such as those encountered during spaceflight and on the International Space Station. It may also allow the identification of novel genetic engineering strategies aimed at improving plant adaptation to the microgravity environment, with potential impact on our ability to improve the genome of other plant species / crops for utilization in bioregenerative life-support systems during spaceflight. A better understanding of plant responses to stress may also have important applications in agriculture for crop production on marginal lands.

New large-scale RNAseq expression data associated with growth under microgravity for previously untested Arabidopsis genotypes will also be generated during this project and submitted to the GeneLab Data System.

This project will allow us to introduce undergraduate students to the idiosyncrasies of scientific research in Space Biology, and also generate novel instructional materials allowing effective teaching of the difficult concepts of gravity perception by plants and their importance for agricultural production on Earth and during spaceflight. Such materials should be usable in K-12, undergraduate, and graduate education, as well as general-public outreach.

Task Progress & Bibliography Information FY2023 
Task Progress: The main objective of Advanced Plant Experiment (APEX-08) is to evaluate the impact of genetic alterations of the putrescine pool in Arabidopsis thaliana on its seedlings’ ability to grow, develop, and respond transcriptionally to the microgravity conditions of the International Space Station (ISS) relative to ground control. To this end, we developed and characterized Arabidopsis thaliana mutant and transgenic lines with alterations in putrescine metabolism including: (1) wild type Col-0; (2) adc1-1 knockout mutant affected in its ability to synthesize Put; (3) Cuao3-1 and cuao3-100cdr7 allelic mutants affected in their ability to catabolize putrescine; (4) transgenic plants over-expressing the Poncirus trifoliata ADC (PtADC) gene involved in putrescine synthesis; and (5) transgenic cuao3 plants over-expressing the AtCuAO3 transgene. We then used these genotypes to evaluate the effect of alterations in the pool of putrescine and/or its catabolites on Arabidopsis thaliana seedling responses to the stress encountered during spaceflight.

Results indicate that seedlings of all genotypes grown under microgravity conditions on the ISS carry longer leaf petioles than those grown in a ground control at NASA Kennedy Space Center (KSC). Furthermore, plants over-expressing the PtADC gene display simplified transcriptional response profiles to spaceflight relative to ground control, with weaker oxidative stress response; whereas those with a knockout mutation in the CuAO3 gene develop more complex response profiles to spaceflight. Furthermore, genes associated with epigenetic regulatory processes such as DNA and histone modifications are differentially regulated between spaceflight and ground control conditions in a genotype- and organ-specific manner. Together, these results suggest increased accumulation of putrescine, and/or putrescine-derived compounds, leads to enhanced tolerance to spaceflight.

Considering this conclusion, we also set up to investigate the ability of plants with the same alterations in the PA metabolic pathway to grow and develop on lunar regolith simulant (LHS-1), a substrate previously suggested to be stressful for plants. The results of preliminary seedling-viability experiments suggest plants over-expressing the PtADC gene also display increased tolerance to lunar regolith simulants. Our results suggest a novel genetic engineering strategy to engineer plants with improved tolerance to spaceflight and/or lunar regolith simulant, with important implications for space exploration programs.

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
Articles in Peer-reviewed Journals Su S-H, Levine HG, Masson PH. "Brachypodium distachyon seedlings display accession-specific morphological and transcriptomic responses to the microgravity environment of the International Space Station." Life. 2023 Feb 23;13(3):626. https://doi.org/10.3390/life13030626 ; PMID: 36983782; PMCID: PMC10058394 , Feb-2023
Awards Su S-H. "University of Wisconsin-Madison Award for Mentoring Undergraduates in Research, Scholarly and Creative Activities. " May-2023
Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2022 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 09/15/2023  
Task Last Updated: 07/14/2022 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Su, Shih-Heng  Ph.D. University of Wisconsin-Madison 
Key Personnel Changes / Previous PI: July 2020 report: Dr. Shih-Heng Su is an Assistant Scientist in the Masson lab who has been contributing to this project since its inception.
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Flight Assignment/Project Notes: NOTE: End date changed to 09/15/2023 per NSSC information (Ed., 7/26/22).

Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station (ISS) and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project will lead to a better understanding of the role of Putrescine and/or its derived signaling products in mitigating the stress associated with growth under microgravity environments such as those encountered during spaceflight and on the International Space Station. It may also allow the identification of novel genetic engineering strategies aimed at improving plant adaptation to the microgravity environment, with potential impact on our ability to improve the genome of other plant species / crops for utilization in bioregenerative life-support systems during spaceflight. A better understanding of plant responses to stress may also have important applications in agriculture for crop production on marginal lands.

New large-scale RNAseq expression data associated with growth under microgravity for previously untested Arabidopsis genotypes will also be generated during this project and submitted to the GeneLab Data System.

This project will allow us to introduce undergraduate students to the idiosyncrasies of scientific research in Space Biology, and also generate novel instructional materials allowing effective teaching of the difficult concepts of gravity perception by plants and their importance for agricultural production on Earth and during spaceflight. Such materials should be usable in K-12, undergraduate, and graduate education, as well as general-public outreach.

Task Progress & Bibliography Information FY2022 
Task Progress: SUMMARY

The main objective of APEX-08 is to evaluate the impact of genetic alterations of the putrescine pool in Arabidopsis thaliana on the seedlings’ ability to grow, develop, and respond transcriptionally to the microgravity conditions of the International Space Station (ISS) relative to ground control. To this end, we previously reported on the development and characterization of Arabidopsis thaliana mutant and transgenic lines with alterations in putrescine metabolism, including: (1) wild type Col-0, (2) adc1-1 mutant affected in its ability to synthesize Put; (3) Cuao3-1 and cuao3-100cdr7 mutants affected in their ability to catabolize putrescine; (4) transgenic plants over-expressing the Poncirus trifoliata ADC (PtADC) gene involved in putrescine synthesis (hereafter named PtADC-OX); and (5) transgenic cuao3 plants over-expressing the AtCuAO3 gene (hereafter named cuao3-1[CuAO3]-OX). These genotypes should allow evaluation of the effect of putrescine-pool alterations on Arabidopsis thaliana responses to the stress encountered under microgravity.

In 2021, we reported on experiments that allowed us to bulk up seed material from these wild type, mutant, and over-expression lines for verification tests as well as for the flight and ground control experiments, and successfully completed both the Science Verification (SVT) and Experimental Verification (EVT) tests at NASA Kennedy Space Center (KSC). Preparation for these tests and their implementation allowed us to optimize the experimental approach and success criteria for APEX-08. Consequently, we were able to implement both the APEX-08 flight experiment on the International Space Station (ISS) and its ground control at KSC in the fall of 2021. As discussed below, both experiments went smoothly, and their results are currently being analyzed.

PROGRESS MADE DURING THE 2021-22 PERIOD

1) Implementation of APEX-08

1.a. Experimental Approach:

The APEX-08 experiment was carried out following the approach we optimized during the SVT (November 2020) and EVT (March 2021). Briefly, 10x10" square Petri dishes were poured with 1/2MS medium containing 0.8% agar at KSC on 8/24/2021. The next day, seeds from each genotype were surface-sterilized and plated on the surface of the medium, in a line, at a density of 50 seeds per plate. Each genotype was represented by 5 plates (5 replicates). Seeded plates were immediately treated with far-red light in the cold room in darkness, then wrapped in aluminum foil and stored at 4°C until handed over to the Multi-Element Integrated Test (MEIT) team for cold storage the next day. Upon transfer into the Dragon CRS-23 capsule, the experiment took off on SpaceX CRS-23 (SpX-23) on 8/29/2021. After reaching the ISS, the plates were unwrapped and inserted into the NASA Vegetable Production System (Veggie) growth unit by astronaut Shane Kimbrough on 9/1/2021, following the blueprint previously established for the SVT. Pictures of each plate were taken before insertion, to make sure seed germination did not occur during takeoff and the plates were not cracked. Germination was then triggered by a 24-h red light treatment. After one day, both green and blue lights were turned on (low setting) and the seedlings were allowed to germinate and grow for an additional 8 days. The temperature, humidity, and CO2 profiles were recorded with HOBO sensors located near the plates on Veggie.

At the end of this growth period, plates were photographed, and the seedlings were harvested into RNAlater within NASA Kennedy Fixation Tubes (KFTs). After 1 day at room temperature, the KFTs were transferred into the -80°C freezer until returned to the ground on 9/30/21.

A ground control (GC) mimicking all aspects of the flight experiment was carried out at KSC, with a 48-h delay. Here again, the 30 plates submitted to this experiment were photographed both at the time of insertion and after the growth period. Seedlings were harvested in RNAlater within KFTs at the end of the experiment, then frozen at -80°C for storage and transported to the Principal Investigator (PI) laboratory for further analysis.

At the end of the experiment, the photographs were used to evaluate the effect of microgravity on seedling growth and morphology relative to ground control, and harvested tissues were used to extract RNA for expression profiling by RNAseq. Preliminary results from these experiments are summarized below.

1.b. Preliminary Results:

1.b.1. Far-red light pretreatment inhibits the germination of plated seeds

To verify the effectiveness of far-red light pretreatment at inhibiting the germination of plated seeds and also ensure that seeds remained attached to the medium and the plates did not crack during takeoff, we analyzed the photographs taken by astronaut Shane Kimbrough at the time of insertion on the ISS, and by the MEIT team for the ground control at KSC. In both cases, there was no evidence of cracked plates, detached or germinated seeds, indicating the experiment could proceed as planned.

1.b.2. A 9-day growth period in Veggie on the ISS (Flight experiment, FL) and on the ground at KSC (ground control, GC) led to high germination rates and production of significant amounts of plant biomass, adequate for initial morphometric analysis as well as RNA extraction and RNAseq analysis.

1.b.2.a. Morphometric Analysis

Analysis of the photographs taken at the end of the growth period for both flight and ground-control groups indicated close to 100% germination for all genotypes tested, although it was not possible to assign a precise germination rate because the seedlings were intertwined at this late stage of growth due to the high-density plating necessary to obtain sufficient material for RNA extraction and subsequent analysis.

In both the flight and ground-control experiments, the seedlings grew well, eventually covering most of the plate surfaces. A very noticeable difference between flight and ground control seedlings of all genotypes was a general tendency for the roots to grow downward in the ground control, and more randomly in the flight experiment.

The high density of seedlings within each plate prevented us from performing a morphometric analysis of organs' growth using the photographs taken at the end of the growth period. However, upon receipt of the fixed and frozen tissue samples from KSC at the end of the experiment, we were able to retrieve a few seedlings from the bulk before RNA extraction, which we flattened on a glass surface and used to evaluate shoot sizes. This analysis was very informative, indicating a significant increase in green-leaf area and petiole sizes for both cotyledons and the first two true leaves in microgravity-exposed samples relative to ground controls. The differences in petiole sizes were significant for all genotypes tested. The cotyledon petiole size ratio between flight and ground control samples, on the other hand, showed no significant differences between genotypes. The latter observation may be explained by the fact that cotyledons are embryonic organs that formed during seed development, before microgravity exposure. Finally, looking at total leaf area, adc1-1 mutant seedlings displayed smaller values under ground-control conditions, but this difference was completely alleviated under flight conditions, suggesting that a decrease in arginine decarboxylase activity in the mutant (likely to result in decreased putrescine synthesis) did not affect its ability to develop biomass relative to wild type under microgravity conditions.

To identify possible border-effects on measured morphological phenotypes, we used ANOVA. Results indicate the existence of only minor effects from the corner positions within Veggie in the flight experiment, which were not recapitulated on the ground.

1.b.2.b. RNA extraction and RNAseq analysis

Half of the fixed tissue materials returned to the lab after harvesting in both the flight experiment on the ISS and the ground control at KSC, were recovered, dissected for separation of shoot and root tissues, and subjected to RNA extraction. Extracted RNAs were then analyzed using a combination of Nanodrop and TapeStation procedures. Overall, RNAs of sufficient quality for library construction and RNAseq analysis were obtained from all spaceflight and ground-control tissues.

After rRNA reduction, these RNA samples were subjected to TruSeq library construction, and sequencing by the Wisconsin Gene Expression Center at the University of Wisconsin Biotechnology Center. Sequence reads are currently being analyzed.

2) Conclusion

The experimental conditions that were optimized during our Science- and Experiment-Verification Tests at KSC last year allowed successful implementation of both our flight-ISS and ground-control experiments. In both cases, all six genotypes displayed excellent germination and growth, meeting the experimental criteria defined in the NASA Environmental Resources Document (ERD). The seedlings of cuao3-100cdr7 grew more slowly than those of the five other lines, as expected from our initial results during the SVT and EVT as well as other experiments in the lab.

The high plant density chosen for APEX-08 prevents careful biometric analysis of plant morphology and development. However, we were able to evaluate the effect of spaceflight on the morphology of the shoots by recovering several seedlings from the fixed and frozen material returned to the lab for RNA extraction and expression analysis, and we analyzed them for total leaf area, cotyledon, and first-two-leaves petiole sizes. Interestingly, all genotypes showed evidence of increased growth under microgravity relative to ground control: The total leaf area was larger in flight-exposed seedlings, in large part because the petioles were longer. For cotyledon petiole size, the ratio between flight-exposed and ground-control plants was similar for all genotypes tested, indicating exposure to the microgravity environment of the ISS had little impact on the development of these embryonic organs. On the other hand, a similar ratio for first-two-leaves petiole length was higher in adc1-1 and Col[CuAO3] over-expressing plants, suggesting a decrease in putrescine levels might facilitate leaf-petiole growth under microgravity. This conclusion seems compatible with the lower response ratio observed in the Col[PtADC1] over-expressing and cdr7 mutant lines.

The RNAlater-fixed and frozen plant materials that had been harvested in both the flight experiment and the ground control were adequate for RNA extraction and RNAseq analysis, meeting the criteria established by the EVT ERD. They were subjected to RNAseq analysis at the Wisconsin Gene Expression Center. Sequence reads are currently being analyzed. They should provide exciting new clues to better understand the impact of both microgravity and alterations in the putrescine metabolic pathway on Arabidopsis seedling growth and stress responses.

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
Abstracts for Journals and Proceedings Su S-H, Moen AD, Groskop RM, Baldwin K, and Masson PH. "Using low-speed 2-dimensional clinorotation to study gravi-sensitivity in Arabidopsis and Brachypodium." 37th Annual Meeting of the American Society for Gravitational and Space Research, Baltimore, MD, November 3-6, 2021.

Abstracts. 37th Annual Meeting of the American Society for Gravitational and Space Research, Baltimore, MD, November 3-6, 2021. , Nov-2021

Abstracts for Journals and Proceedings Masson PH, Su S-H, Moen AD, Groskop RM, Baldwin K. "Using low-speed 2-dimensional clinorotation to study gravi-sensitivity in Arabidopsis and Brachypodium." 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. Session F1-1. , Jul-2022

Abstracts for Journals and Proceedings Su S-H, Masson PH. "Can you grow plants in space? – Using Brachypodium and Arabidopsis to investigate plant adaptation to spaceflight. " Outreach seminar, National Space Organization, HsinChu City, Taiwan, 2022.

Abstracts. Outreach seminar, National Space Organization, HsinChu City, Taiwan, 2022. , Jan-2022

Articles in Peer-reviewed Journals Gibbs NM, Su SH, Masson PH. "Application of cadaverine to inhibit biotin biosynthesis in plants." Bio Protoc. 2022 Apr 20;12(8):e4389. http://dx.doi.org/10.21769/BioProtoc.4389 ; PMID: 35800104; PMCID: PMC9081473 , Apr-2022
Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2021 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 09/15/2022  
Task Last Updated: 07/13/2021 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Su, Shih-Heng  Ph.D. University of Wisconsin-Madison 
Key Personnel Changes / Previous PI: July 2020 report: Dr. Shih-Heng Su is an Assistant Scientist in the Masson lab who has been contributing to this project since its inception.
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station (ISS) and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project will lead to a better understanding of the role of Putrescine and/or its derived signaling products in mitigating the stress associated with growth under microgravity environments such as those encountered during spaceflight and on the International Space Station. It may also allow the identification of novel genetic engineering strategies aimed at improving plant adaptation to the microgravity environment, with potential impact on our ability to improve the genome of other plant species / crops for utilization in bioregenerative life-support systems during spaceflight. A better understanding of plant responses to stress may also have important applications in agriculture for crop production on marginal lands.

New large-scale RNAseq expression data associated with growth under microgravity for previously untested Arabidopsis genotypes will also be generated during this project and submitted to the GeneLab Data System.

This project will allow us to introduce undergraduate students to the idiosyncrasies of scientific research in Space Biology, and also generate novel instructional materials allowing effective teaching of the difficult concepts of gravity perception by plants and their importance for agricultural production on Earth and during spaceflight. Such materials should be usable in K-12, undergraduate, and graduate education, as well as general-public outreach.

Task Progress & Bibliography Information FY2021 
Task Progress: The main objective of Advanced Plant Experiment (APEX)-08 is to evaluate the impact of genetic alterations of the putrescine pool in Arabidopsis thaliana on the seedlings’ ability to grow, develop, and respond transcriptionally to the microgravity conditions of the International Space Station (ISS) relative to ground control. This experiment should allow an initial evaluation of a possible effect of putrescine and derived compounds on a plant’s ability to mitigate the stress associated with exposure to the microgravity environment of ISS.

Last year, we reported on the development and initial characterization of Arabidopsis thaliana mutant and transgenic lines with alterations in genes that encode enzymes involved in putrescine metabolism. These lines include: (1) wild type Col-0, (2) adc1-1 mutant affected in its ability to synthesize Put; (3) cuao3-1 and cuao3-100cdr7 mutants affected in their ability to catabolize putrescine; (4) transgenic plants over-expressing the Poncirus trifoliata ADC (PtADC) gene; and (5) transgenic cuao3 plants over-expressing the AtCuAO3 gene (cuao3[CuAO3]). These genotypes were developed because they should allow evaluation of the effect of putrescine-pool alterations on Arabidopsis thaliana responses to the stress encountered under microgravity. These experiments should also allow us to investigate a possible contribution of putrescine degradation products such as GABA and hydrogen peroxide in these responses.

This year, we completed our molecular analysis of these Arabidopsis lines, evaluated the effect of these mutations and transgenes on expression of the targeted genes, propagated each line to generate enough seeds for utilization in the Science and Experiment Verification Tests (SVT and EVT) and in the flight experiment. We also investigated and demonstrated the effectiveness of far-red light pretreatments on preventing seed germination after plating on agar-based media, thereby maintaining seed dormancy for all six genotypes under investigation during vehicle loading, takeoff, and transport to ISS. Germination can then be triggered by exposure to light upon transfer into the VEGGIE growth chamber. We completed the SVT and EVT at Kennedy Space Center (KSC). The results of our SVT were used to refine the experimental approach and success criteria for APEX-08, allowing us to successfully complete our EVT. Therefore, APEX-08 is now ready for implementation on the ISS. It has been tentatively scheduled for takeoff on Space X-23 in August 2021.

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
Articles in Peer-reviewed Journals Gibbs NM, Su S-H, Lopez-Nieves S, Mann S, Alban C, Maeda HA, Masson PH. "Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis." Plant J. 2021 Sep;107(5):1283-98. Epub 2021 July 21. https://doi.org/10.1111/tpj.15417 ; PMID: 34250670 , Sep-2021
Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2020 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 09/15/2022  
Task Last Updated: 07/17/2020 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Key Personnel Changes / Previous PI: July 2020 report: Dr. Shih-Heng Su is an Assistant Scientist in the Masson lab who has been contributing to this project since its inception.
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station (ISS) and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project will lead to a better understanding of the role of Putrescine and/or its derived signaling products in mitigating the stress associated with growth under microgravity environments such as those encountered during spaceflight and on the International Space Station. It may also allow the identification of novel genetic engineering strategies aimed at improving plant adaptation to the microgravity environment, with potential impact on our ability to improve the genome of other plant species / crops for utilization in bioregenerative life-support systems during spaceflight. A better understanding of plant responses to stress may also have important applications in agriculture for crop production on marginal lands.

New large-scale RNAseq expression data associated with growth under microgravity for previously untested Arabidopsis genotypes will also be generated during this project and submitted to the GeneLab Data System.

This project will allow us to introduce undergraduate students to the idiosyncrasies of scientific research in Space Biology, and also generate novel instructional materials allowing effective teaching of the difficult concepts of gravity perception by plants and their importance for agricultural production on Earth and during spaceflight. Such materials should be usable in K-12, undergraduate, and graduate education, as well as general-public outreach.

Task Progress & Bibliography Information FY2020 
Task Progress: Plants grown under microgravity conditions display morphological and molecular phenotypes that are compatible with stress exposure. Interestingly, on Earth plants exposed to stress usually accumulate large amounts of polyamines, including putrescine and its derivatives spermidine and spermine, and these compounds typically contribute to stress mitigation [1]. However, analyses of plant transcriptional responses to microgravity suggest a failure to activate polyamine synthesis and response pathways. Considering these observations, we suggested that a failure for plants to activate polyamine synthesis and response pathways under microgravity conditions may be responsible, at least in part, for the altered growth and stress-like phenotypes displayed by microgravity-exposed plants. Advanced Plant Experiment (APEX)-06’s main objective is to investigate the ability of wild type, mutant, and transgenic Arabidopsis plants with modified abilities to either accumulate or oxidize putrescine, to grow, develop and respond transcriptionally to the microgravity conditions of the International Space Station, and compare these growth and molecular responses to those of ground-control plants.

The following Arabidopsis thaliana genotypes will be used in these experiments: (1) wild type Col-0, (2) adc1-1 mutant affected in its ability to synthesize Put, (3) Cuao3-1 and cuao3-100cdr7 mutants affected in their ability to catabolize putrescine, (4) transgenic plants over-expressing the Poncirus trifoliata ADC (PtADC) gene (hereafter named PtADC-OX), and (5) transgenic cuao3-1 plants over-expressing the AtCuAO3 gene (hereafter named cuao3-1[CuAO3]-OX). Utilization of these genotypes should allow us to evaluate the effect of altering the pool of putrescine within Arabidopsis thaliana seedlings on plant responses to the stress encountered under microgravity, and also investigate a possible contribution of putrescine degradation products such as GABA and hydrogen peroxide, to plant stress mitigation under microgravity.

Last year, we generated some of the genetic material needed for these experiments, and also bulked up the corresponding seed stocks to generate enough material for the Science and Experimental Verification Tests (SVT and EVT) at Kennedy Space Center, and for the flight experiment and its ground control.

1) Seeds from wild-type Col-0, mutant cuao3-1, cuao3-100cdr7 and adc1-1, and transgenic cuao3-1[CuAO3]-OX, had previously been generated in the lab by former graduate student Amy Jancewicz [2]. We germinated and grew multiple plants for each one of these genotypes, and self-fertilized them, allowing the production of enough seeds from each genotype to carry out the SVT, EVT, flight experiment and its associated ground control. We also used PCR analysis to verify the genotypes of these plants. Furthermore, leaf material was collected from each plant and stored at -80°C for subsequent RNA extraction and expression studies. Current and future work is aimed at verifying the phenotype associated with each line, examining the level of targeted gene/transgene expression in these plants relative to wild type, and identifying independent transgenic lines that over-express the corresponding transgene relative to wild type.

2) We were also planning on using seeds from a transgenic Arabidopsis thaliana line that was previously shown to over-express the Poncirus trifoliata ADC (PtADC) gene, accumulate putrescine in their tissues, and display increased tolerance to abiotic stresses [3]. Unfortunately, we subsequently learned that this material had been lost by the lab that generated it. Therefore, we built a new construct aimed at over-expressing the PtADC transgene in Arabidopsis thaliana. A cDNA encoding the PtADC open reading frame was synthesized, fused to the CaMV 35S promoter and nos terminator sequences, and introduced into the pAGM4673 level-2 destination vector, along with a spectinomycin-resistance selection cassette, using the Golden Gate technology [4]. A similar construct carrying an internally deleted version of the CaMV35Sp::PtADC-Nost cassette (with an internal 0.6 kbp BsaI segment deleted from the PtADC open-reading-frame) was also constructed for use as a negative control (hereafter named PtADC-OX). After verification by sequencing, these constructs were introduced into wild type Col-0, adc1-1 and adc2-1 mutant plants using the Agrobacterium tumefaciens-mediated floral-dip in planta transformation approach [5]. Transformed progeny were selected on spectinomycin-containing 0.8% agar-based 1/2MS media, propagated, and self-fertilized. Their progeny were again selected on spectinomycin-containing media, and propagated. Leaf samples were harvested from individual transformants, and frozen in liquid nitrogen. RNA is being extracted from these samples, which will be used to evaluate transgene expression levels in independent transgenic lines, using RT-qPCR approaches. Interestingly, some of these PtADC-OX transgenic plants displayed distinct levels of growth and morphological phenotypes depending on the line analyzed, including shorter organs, spiraling leaves, and partial to complete sterility. These phenotypes were not observed on plants transformed with the internally deleted version of PtADC (PtADC-OX). The variable nature of these phenotypes between independently isolated lines suggests that they might derive from variations in levels of transgene over-expression due to position effects. We are testing this hypothesis by investigating the level of transgene expression in leaf tissues from these plants relative to wild type, using RT-qPCR approaches. Growth and morphological phenotypes will be compared between lines showing distinct levels of transgene over-expression. Additional experiments will also allow evaluation of plant sensitivity to abiotic stresses such as osmotic, salt, drought, and hypoxia.

We expect higher levels of transgene expression in plants displaying the strongest phenotypes, and lower levels in normal looking plants [6]. We also expect increased tolerance to at least some of these stresses for over-expressing transgenic plants relative to wild type, and increased sensitivity to stress for the corresponding knockout mutant plants.

These experiments will allow us to select transgenic plants that appear morphologically normal, but still display increased transgene expression levels relative to wild type, along with altered tolerance to abiotic stresses, for use in our SVT, EVT, and spaceflight experiments.

This project was spearheaded by Dr. Shih-Heng Su, helped by undergraduate student Tyler Serie Anne Ganser for plant cultivation and seed bulking. The Covid-19 lockdown interfered with progress in this project, starting mid-March 2020 and lasting until the end of June, 2020. However, we were still allowed to continue bulking seeds from our wild type, mutant and transgenic plants during this period, a process that allowed us to generate enough material to test our lines and, in the near future, carry out the SVT, EVT, flight and ground control experiments. Plant tissues were also harvested during this period, which we are currently using to investigate target gene expression levels in our lines.

Cited Literature

[1] Jancewicz, A., Gibbs, N. & Masson, P. Cadaverine's functional role in plant development and environmental response. Front Plant Sci 7, 1-8, doi:10.3389/fpls.2016.00870 (2016).

[2] Jancewicz, A. A Genetic Study of Cadaverine Response Reveals Crosstalk Between Cadaverine and Putrescine Pathways in Arabidopsis thaliana PhD thesis, University of Wisconsin-Madison, (2017).

[3] Wang, J. et al. An arginine decarboxylase gene PtADC from Poncirus trifoliata confers abiotic stress tolerance and promotes primary root growth in Arabidopsis. J Exp Bot 62, 2899-2914, doi:10.1093/jxb/erq463 (2011).

[4] Engler, C. et al. A golden gate modular cloning toolbox for plants. ACS Synth Biol 3, 839-843, doi:10.1021/sb4001504 (2014).

[5] Bent, A. Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiol 124, 1540-1547 (2000).

[6] Alcázar, R., García-Martinez, J., Cuevas, J., Tiburcio, A. & Altabella, T. Overexpression of ADC2 in Arabidopsis induces dwarfism and late-flowering through GA deficiency. Plant J 43, 425-436, doi:10.1111/j.1365-313X.2005.02465.x (2005).

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
Articles in Peer-reviewed Journals Su S-H, Keith MA, Masson PH. "Gravity signaling in flowering plant roots." Plants. 2020 Oct;9(10):1290. [Published online: 29 September 2020.] https://doi.org/10.3390/plants9101290 ; PMID: 33003550; PMCID: PMC7601833 , Oct-2020
Project Title:  Can Polyamines Mitigate Plant Stress Response under Microgravity Conditions? Reduce
Images: icon  Fiscal Year: FY 2019 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Cell & Molecular Biology   | Plant Biology  
Start Date: 09/16/2019  
End Date: 09/15/2022  
Task Last Updated: 09/11/2019 
Download Task Book report in PDF pdf
Principal Investigator/Affiliation:   Masson, Patrick H. Ph.D. / University of Wisconsin Madison 
Address:  Laboratory of Genetics (Room 3262) 
425G Henry Mall 
Madison , WI 53706-1580 
Email: phmasson@wisc.edu 
Phone: 608-265-2312  
Congressional District:
Web: https://masson.genetics.wisc.edu/  
Organization Type: UNIVERSITY 
Organization Name: University of Wisconsin Madison 
Joint Agency:  
Comments:  
Project Information: Grant/Contract No. 80NSSC19K1483 
Responsible Center: NASA KSC 
Grant Monitor: Zhang, Ye  
Center Contact: 321-861-3253 
Ye.Zhang-1@nasa.gov 
Unique ID: 12526 
Solicitation / Funding Source: 2018 Space Biology (ROSBio) NNH18ZTT001N-FG. App B: Flight and Ground Space Biology Research 
Grant/Contract No.: 80NSSC19K1483 
Project Type: Flight,Ground 
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) Plant Biology
Space Biology Cross-Element Discipline: None
Space Biology Special Category: (1) Bioregenerative Life Support
Task Description: Space exploration requires the design and implementation of bioregenerative life-support systems capable of maintaining breathable air, cleaning water, and producing food and fiber during space missions. Plants are expected to play key roles in these life-support systems. Therefore, it is important to understand the effects the microgravity environment has on plants to allow the design and development of better life-support systems for space exploration. Multiple studies have demonstrated that plants grown under microgravity display signs of stress, including altered morphology and expression profiles expected for stress response. Therefore, the development of mitigation strategies aimed at alleviating such stress will be important. Previous studies by multiple research teams including ours have demonstrated a role for polyamines in stress mitigation in plants. Polyamines are a group of compounds whose abundance increases in plants under stress. These signaling molecules have also been shown to contribute to stress mitigation in ground-based experiments. Therefore, we hypothesize that polyamines might also contribute to stress mitigation under microgravity, and propose to test this hypothesis by investigating the growth and expression profiles of Arabidopsis thaliana seedlings engineered to modify the pool of putrescine (one of these polyamines) accumulating in their tissues. Specific objectives will include: 1) Comparing the growth of Arabidopsis thaliana seedlings with genetic alterations affecting the pool of putrescine to that of wild type, within the VEGGIE hardware, both under microgravity on the International Space Station and on the ground (1-g control), and 2) Use RNAseq approaches to investigate the effect of microgravity relative to 1-g ground control on the transcriptomes of wild type and mutant seedlings with alterations in their putrescine pool. These investigations should lead to a better understanding of the roles played by polyamines in stress mitigation under microgravity. It may identify novel genetic engineering methods that improve plant growth under microgravity, thereby allowing development of optimized bioregenerative life-support systems for long-term space exploration missions. This project may also have important applications in agriculture for crop production on marginal lands. Finally, our experiments will yield large sets of transcriptomic data from previously uncharacterized genotypes, which will be added to the GeneLab dataset.

Research Impact/Earth Benefits: This project may also have important applications in agriculture for crop production on marginal lands.

Task Progress & Bibliography Information FY2019 
Task Progress: New project for FY2019.

Bibliography: Description: (Last Updated: 07/03/2024) 

Show Cumulative Bibliography
 
 None in FY 2019