NOTE: End date changed to 3/15/2022 per NSSC information (Ed., 3/16/21) NOTE: Extended to 3/15/2021 per NSSC information (Ed., 12/16/19)

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

NOTE: End date changed to 3/15/2022 per NSSC information (Ed., 3/16/21) NOTE: Extended to 3/15/2021 per NSSC information (Ed., 12/16/19)

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

In April and May 2019, 3 undergraduate students identified T-DNA insertion lines available for approximately half of the contrast analysis target list, and they spent the ensuing 3 months testing about 35 lines for gravity phenotypes. These data were presented by undergraduate students at ASGSR 2019 in Denver, CO at the end of November [Ed. Note: 2019 Annual Conference of the American Society for Gravitational and Space Research] and already indicate numerous genes with hitherto unreported gravity phenotypes, greatly expanding our understanding of this response pathway. The pandemic prevented us from continuing this screen in the summer of 2020, but we continued the work in summer 2021.

We have started the analysis of the large dataset generated by RNA sequencing (RNA-seq) of the samples grown for 36 h under altered g conditions on the ISS using the European Modular Cultivation System (EMCS) centrifuge. At this time, we have carried out exploratory data analysis in order to begin to understand the most fruitful approaches and comparisons to choose for the identification of differentially expressed genes and pathways. One key observation is the dramatic difference in the overall regulation of gene expression between wild-type (WT) and starchless (pgm-1) mutants. Plotting a heatmap of expression of the 1000 most variable genes reveals this difference in pattern, with WT seedling roots showing much more consistent regulation than starchless mutants. We are currently analyzing all 384 transcriptomes compared amongst each other to infer patterns of plant transcriptional regulation that may be responsive to the gravity cue as a continuum.

NOTE: End date changed to 3/15/2022 per NSSC information (Ed., 3/16/21) NOTE: Extended to 3/15/2021 per NSSC information (Ed., 12/16/19)

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

Samples returned from flight, where they were exposed for 36 h to specific artificial gravity treatments using the European Modular Cultivation System (EMCS) centrifuge, were processed to extract RNA. Seedlings were dissected into 4 organs (cotyledons, hypocotyls, mature roots, and root tips) and RNA was extracted separately for each organ across a range of 12 gravity treatments for both WT and starchless (pgm-1) mutants. Each organ-gravity-genotype sample was replicated 4 times in the design to maximize statistical robustness. After confirming the quality of RNA samples via Bioanalyzer chip, they were shipped to a core facility for consistent library preparation using the Takara low-input library kit. Libraries were sequenced on an Illumina NovaSeq 6000 using paired-end, 150 bp reads. Both the library preparations and the sequencing were delayed due to laboratory shutdowns, with the final flight reads completed in November 2020.

Due to the delays in completing the sequencing process, we have only recently started to analyze the RNA-seq data, which totals 400 sample libraries and approximately 2 TB of data. We are coordinating the upload of the raw read files to NASA’s GeneLab platform for widespread availability of the data and analysis with the community. Fortunately, our collaborators, Sarah Wyatt and Al Meyers, have built and tested the analysis pipeline to be used for processing the data, using our previous pilot data set for development and optimization. We anticipate being able to complete the first analysis of these data by Summer 2021. These data will allow us to directly probe the effect of gravity as a continuum on plant seedling growth and establishment from a minimum of 0.006 g up to 1.0 g, representing the most complete and systematic look at the effect of this key variable on gene expression to date.

In addition to the work on preparing and sequencing flight samples, we also made progress on identifying novel components of the gravity signaling pathway in roots. We had previously identified a list of 124 genes showing contrasting expression patterns between WT and pgm-1 mutants under vertical compared to gravistimulated conditions. As a first step toward testing the roles of some of these genes in gravity response, students tested about 35 T-DNA lines with insertions in some of these genes. While the pandemic and ensuing shutdown precluded students from working in the lab over the summer, students made progress in Fall 2020 toward confirming the genotypes and transcript abundance in these mutant lines using RT-qPCR.

NOTE: Extended to 3/15/2021 per NSSC information (Ed., 12/16/19)

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

The Plant Gravity Perception (PGP) payload was launched on SpaceX CRS13 in December of 2017 and operations were carried out January 26 - April 12, 2018. The duration of the flight experiments was increased substantially due to the unpredictable behavior of the European Modular Cultivation System (EMCS) facility on the ISS, which had problems powering the hardware throughout all four runs. Despite this difficulty the main objectives of PGP were accomplished with a minimum of science loss thanks to the tremendous effort of all team members. Germination rates for both wild-type and starchless mutants was approximately 84% for the spaceflight experiment, yielding more than sufficient quantities of seedlings for both image analysis of the responses to fractional gravity and light as well as for isolation of RNA for RNAseq analysis.

Seedlings on orbit demonstrated clear and consistent responses to unilateral blue light stimulation in microgravity conditions (no fractional g treatment), with roots orienting away from the direction of light stimulation as expected. The response of seedling roots to the ensuing fractional gravity treatment showed a strong dependence upon the genotype of the plant, with wild-type roots having a much greater sensitivity to fractional g treatments than starchless mutants. This was also predicted, but the magnitude of difference was unknown and will inform our interpretation of the molecular-level differences we anticipate from the RNAseq data.

In addition to the flight experiments, we also successfully carried out a ground-based pilot RNAseq experiment in preparation for processing the flight tissue samples, which returned in May 2018. We designed an experiment using wild-type and pgm-1 starchless mutants in which we collected tissue for processing from vertically-growing seedlings and seedlings receiving a 90 degree reorientation 10 min prior to freezing and tissue harvesting. Following freezing, all seedlings were dissected into root tips, root bases, hypocotyls, and cotyledons and RNA isolation was carried out on 4-7 seedling tissues per type with 4 replicates of each treatment and genotype. RNA samples were used for cDNA synthesis and library preparation using a low-input cDNA library kit, and libraries were sequenced on an Illumina NovaSeq 6000. We have completed differential gene expression analysis now, including a contrast analysis, which resulted in the identification of more than 100 genes with contrasting expression patterns across treatments and genotypes. This ground-based data set will form a critical resource for comparison against our flight data set as well as providing a rich resource of candidate genes to be tested for a role in gravity perception or signal transduction.

As a first step toward testing the function of these genes in the gravity response pathway, we obtained T-DNA insertional mutants in 35 of these genes showing contrasting expression and carried out a series of phenotyping experiments. In addition to the standard root gravitropism assay, we developed a new assay based on our ROTATO image analysis and feedback rotation system that stimulates the root by 90 deg, then continues to rotate the plate at a constant rate of rotation. This technique requires the root to continue expressing differential growth, something that wild-type roots do with ease, but roots defective in gravity perception or response fail to maintain. This combination of assays has identified 10 of the 35 mutants tested thus far to show some degree of gravity-related phenotype. We believe this approach holds promise to identify and map more of the gravity response machinery, and we look forward to building on these data and those that emerge from the differential expression data from seedlings exposed to fractional amounts of g.

NOTE: Extended to 3/15/2021 per NSSC information (Ed., 12/16/19)

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

The Plant Gravity Perception (PGP) payload was launched on SpaceX CRS13 in December of 2017 and operations were carried out January 26 - April 12, 2018. The duration of the flight experiments was increased substantially due to the unpredictable behavior of the EMCS facility on the ISS, which had problems powering the hardware throughout all four runs. Despite this difficulty the main objectives of PGP were accomplished with a minimum of science loss thanks to the tremendous effort of all team members. Germination rates for both wild-type and starchless mutants was approximately 84% for the spaceflight experiment, yielding more than sufficient quantities of seedlings for both image analysis of the responses to fractional gravity and light as well as for isolation of RNA for RNAseq analysis.

Seedlings on orbit demonstrated clear and consistent responses to unilateral blue light stimulation in microgravity conditions (no fractional g treatment), with roots orienting away from the direction of light stimulation as expected. The response of seedling roots to the ensuing fractional gravity treatment showed a strong dependence upon the genotype of the plant, with wild-type roots having a much greater sensitivity to fractional g treatments than starchless mutants. This was also predicted, but the magnitude of difference was unknown and will inform our interpretation of the molecular-level differences we anticipate from the RNAseq data.

In addition to the flight experiments, we also successfully carried out a ground-based pilot RNAseq experiment in preparation for processing the flight tissue samples, which returned in May 2018. We designed an experiment using wild-type and pgm-1 starchless mutants in which we collected tissue for processing from vertically-growing seedlings and seedlings receiving a 90 degree reorientation 10 min prior to freezing and tissue harvesting. Following freezing, all seedlings were dissected into root tips, root bases, hypocotyls, and cotyledons and RNA isolation was carried out on 4-7 seedling tissues per type with 4 replicates of each treatment and genotype. RNA samples were used for cDNA synthesis and library preparation using a low-input cDNA library kit, and libraries were sequenced on an Illumina NovaSeq 6000. We have carried out quality checks on the reads produced and confirmed the successful sequencing of each sample, and we are now in the process of carrying out differential gene expression analysis. This ground-based data set will form a critical resource for comparison against our flight data set, on which we have recently begun tissue extractions for RNAseq analysis later this year.

NOTE: Extended to 3/15/2020 per NSSC information (Ed., 12/17/18)

NOTE: Extended to 3/15/2019 per NSSC information (Ed., 12/18/17)

With regards to hardware, operations and schedules, the Plant Gravity Perception OVT was considered a success. The functionality of several EMCS operations-related procedures could be verified, e.g., EMCS schedules, EMCS command procedures. Some of the crew procedures were also verified during the EC insertion, EC removal, and sample processing.

Following the successful conclusion of both of the tests described above, on 3 October 2017, a Science Readiness Review was held at ARC. The goal of this review was to obtain concurrence from the NASA ARC Space Biology Division and approval from the NASA HQ Space Life and Physical Sciences Research and Applications (SLPSRA) Division. The results of the Experiment Verification Tests and the OVT were discussed. PGP was permitted to move forward to flight.

The focus of ground experiments this year has been on collecting additional gravitropism data under conditions that more closely mimic the flight with regard to lighting. Our preliminary results show a significant effect of light on the rate of gravitropic response in roots.

The non-flight objective of this project is to characterize mutants in other potential non-statolith components of the gravity perception machinery in plants. We have continued this work this year, focusing on mutants in various components of mechanoperception pathways in plants.

This year we also proposed an augmentation to our original proposal to GeneLab, with the objective of returning the flight plant samples for gene expression analysis. This augmentation proposal, formally submitted in August 2017, was approved for funding. Thus, we began working on the pilot project for this part of the project, aimed at determining the minimum sample requirements to extract usable RNA for RNA-Seq analysis.

This year, which was our second year of funding for this project, we continued to define our experimental plan for our spaceflight experiment. We resolved the last of a number of questions that needed to be answered before our project can proceed toward flight implementation. We also began building a data set of gravity responses with plants growing under the same conditions as the flight experiments.

We finished the last of our preliminary experiments, in which we determined the germination rates of seeds stored in flight conditions for many months, so we can determine how long we can safely store our experiment before launch and while onboard the Space Station before our seeds begin to get too old to germinate and grow normally. We found that seeds attached to flight membranes and stored for even 8 months remain highly viable, with germination rates at or above 90%, as long as the seeds were not glued with an excessive amount of guar gum. The excess gum dramatically reduced germination rates to below 50%, revealing an important parameter in properly setting up the experiment prior to launch.

We carried out a Science Verification Test this year too, the purpose of which is to demonstrate that the plants could grow and respond as required in the same hardware used for the flight experiment. We performed this experiment as much like the flight experiment as possible, and we found that the germination, growth, and response to directional light treatments all fell within the “Excellent” range as established previously for each of these parameters. We also began planning for an Experiment Verification Test, which will take place in a growth chamber that mimics the atmosphere and lighting conditions available on the Space Station.

In addition to these experiments to prepare for flight, we also started studying the gravity response pathway in a number of other mutants, to test whether some of these mutations might give insight into the non-statolith gravity sensing system. To perform these experiments, we use a special device that we made just for measuring gravity responses in plants and that lets us hold the plant very still in a specific position relative to gravity. We are carrying out these experiments in the lab on a continuous basis, looking at many different mutants for clues to gravity sensing in plants. Our homemade device is approaching the end of its useful life, so this year we began work to update and modernize this instrument using off-the-shelf electronic components rather than the previous, custom-made components.

This year, which was our first year of funding for this project, we started to define and plan our spaceflight experiment. We identified a number of questions that need to be answered before our project can be scheduled for flight. We spent most of our time working on designing experiments to answer these questions.

We confirmed that the plants we plan to use as our statolith mutants are the correct plants that truly lack statoliths and began a process of continuously producing fresh seeds for the flight experiment. We compared methods of seed surface sterilization to ensure that our seeds do not carry any contamination while also protecting the embryos from damage. We tested our mutants and normal seedlings under environmental conditions like they will experience on flight, including elevated carbon dioxide levels and various nutrient mixtures to identify the best way to conduct our experiments. We tested our seedlings for growth rates at various stages of development on the special membranes we will use to grow them on flight and compared this with seedling growth on agar, our usual method of growing seedlings. We confirmed that our seedlings will respond to the environmental cues we plan to use in the flight experiment to orient the growth of seedlings in the absence of gravity. We are finishing the last of our preparatory experiments now, in which we are measuring the germination rates of seeds stored in flight conditions for many months, so we can determine how long we can safely store our experiment before launch and while onboard the Space Station before our seeds begin to get too old to germinate and grow normally.

In addition to these experiments to prepare for flight, we also started studying the gravity response pathway in a number of other mutants, to test whether some of these mutations might give insight into the non-statolith gravity sensing system. To perform these experiments, we use a special device that we made just for measuring gravity responses in plants and that lets us hold the plant very still in a specific position relative to gravity. We are carrying out these experiments in the lab on a continuous basis, looking at many different mutants for clues to gravity sensing in plants.