NOTE: Extended to 12/31/2019 per F. Hernandez/ARC (Ed., 5/6/19)

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

Although the uneven growth and germination of seedlings during the Plant RNA Regulation (PRR) spaceflight limited our ability to accomplish our originally proposed objectives, we have nevertheless been successful in carrying out transcriptional profiling of both root and shoot samples. In addition, we were able to also profile sRNAs in shoot tissue and we identified 3 miRNAs that appear to specifically regulate genes encoding proteins with chloroplastic functions. Transcriptional profiling and comparison with our previous spaceflight experiment has revealed consistent patterns of regulation between the two experiments. As reported by other groups, we detected cell wall associated genes to be downregulated in micro g, probably due to the reduced mechanical load. Our results also identify the chloroplast (and chloroplast related functions) as being highly sensitive to the spaceflight environment. Surprisingly, our work has uncovered significant downregulation in micro g of genes involved in stress responses, a result which is counter to some published reports, which compared spaceflight to ground controls. This highlights the importance of having a “1g” control in space, which enabled us to identify primary effects of micro g from overall spaceflight related responses.

ANNUAL REPORTING AS OF SEPTEMBER 2020

1. The Principal Investigator (PI) and graduate student attended ASGSR (American Society for Gravitational & Space Research) in November 2019 in Denver CO. The PI was selected to present a short talk describing this work entitled “Conserved plant transcriptional responses to microgravity from two consecutive spaceflight experiments.”

2. Given the success with the shoot samples, we went ahead and processed all of the root samples. We isolated RNA for root samples representing the 5 different fractional g levels within the ‘1g” rotor as well as micro g from day 4 and day 6 and day 6 ground controls.

3. The RNA samples were submitted for library preparation and NextGen Sequencing to be carried out “in house” at the Genomics Science Laboratory at North Carolina State University (NCSU) in December 2019. Due to their back log, the facility had started the quality check on the samples in March 2020 right before research activities were severely impacted due to the virus pandemic.

4. During the period of March to July, we were under reduced mandatory personnel only operation and therefore were unable to carry out much lab work. During this time the PI worked on data compilation and analysis.

5. Since July 2020 when normal operations were resumed at the Genomic Sciences Laboratory (GSL) we have just received the raw data for the root samples. We are currently carrying out the initial data processing and analysis. We anticipate that this work will be completed within the next few months.

NOTE: Extended to 12/31/2019 per F. Hernandez/ARC (Ed., 5/6/19)

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

1. The Principal Investigator (PI) and graduate student Eric Land attended the ASGSR (American Society for Gravitational & Space Research) conference in Nov 2018. The PI presented a talk entitled “Uncovering transcriptional responses to fractional g in Arabidopsis roots” and the graduate student presented a poster entitled “Transcriptional Regulation of Seedling Development in Microgravity.” The PI served as a judge for the student poster competition.

2. We received the RNA and small RNA-Seq data from Novogene for the Plant RNA Regulation (PRR) shoot samples. We had 3-4 replicate samples for RNA and 3 replicate samples for sRNA, for "1g" in space, microgravity, and ground control for 6 day old samples and “1g” in space and microgravity for 4 day old samples. Overall, the sequence data was good and we have several interesting findings. A comparison of transcriptional profiles from PRR with our previous spaceflight experiment Plant Signaling, shows a highly significant overlap of genes differentially expressed in microgravity between the two experiments. In addition, we analyzed the sRNA landscape of the 1g and micro g shoot samples from PRR. We identified miRNAs that are upregulated in microgravity while several of their corresponding target genes were found to be downregulated in microgravity.

3. We deposited root and shoot RNA-seq data from Plant Signaling to GeneLab.

4. The PI collaborated with Dr. Sarah Wyatt of Ohio University and jointly submitted a proposal for spaceflight research entitled “ Spaceflight alters post-transcriptional regulation,” which was selected for funding in Oct 2019.

5. The PI and graduate student are scheduled to attend the 2019 ASGSR conference. The PI has been selected for a short talk for her abstract entitled “Conserved plant transcriptional responses to microgravity from two consecutive spaceflight experiments.”

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

1. Although a reflight was considered, issues deemed this not feasible; therefore, we have focused on recovering the best science from existing samples.

2. The Principal Investigator (PI) attended ASGSR (American Society for Gravitational & Space Research) conference in October 2017 and served as a judge for the student poster competition.

3. The PI is a participant in the GeneLab AWG plant group. She attended monthly teleconferences and attended the AWG workshop in Florida in April.

4. The graduate student Eric Land was successful in obtaining an NC Space Grant award for research during the summer. He has worked on designing a 2D-clinostat using 3D printing for follow up ground-based studies on transcriptional changes.

5. All Plant RNA Regulation (PRR) flight samples have been harvested and processed for RNA. We are optimizing RNA isolation protocols to ensure retention of sRNA species. Based on sample recovery we have determined that we have sufficient shoot RNA to carry out both RNA and sRNA profiling. Samples will be analyzed by RNAseq and sRNAseq.

6. The PI and graduate student will attend the ASGSR meeting in Oct/Nov 2018 and present their research.

2. A detailed analysis of germination and growth was carried out based on images obtained during PRR. These results indicated that overall germination and growth was reduced with high variability between seed cassettes. Ground testing with flight-build seed and materials showed that the seed stock and flight build were good; the cause of the anomaly is currently unknown.

3. Sample Return via Space-X 10 in April 2017.

2. Ground tests have been completed for seed storage with hydration at 2, 4, and 6 months after plating. Excellent germination and growth have been observed.

3. The Plant RNA Regulation (PRR) experiment passed the flight readiness review in July and was deployed to the International Space Station (ISS) on Space-X9, which launched on July 18, 2016. It is anticipated that the experiment will be conducted in Aug/Sept of 2016.

The major milestones are as follows:

1. Research associate Eric S. Land was recruited to work on this project.

2. The PI team has carried out several ground studies to select new seed stock with good germination and establish the time line for the experiment. Studies have been underway to test the experimental conditions including day length and CO2 concentration as well as long term storage and viability of the seed. 3. The PI and research associate visited NASA Ames Research Center in May 2015. The purpose of the visit was two-fold. The first was to carry out an experiment verification test (EVT) in the ERM-2 module at NASA Ames. The EVT is a ground-based simulation of the conditions of the experiment in space, and in particular the ERM-2 allowed for simulating the lighting, temperature, day length, and CO2 concentration as the flight experiment. The EVT was performed in the ERM-2 module from May 12-18, 2015. Excellent germination and growth of the seedling were confirmed through imaging of the specimens during and at the end of the test. The second objective was to sterilize and mount the seeds and prepare 20 seed cassettes for the Operations Verification Test (OVT) to be performed at N-USOC (Norwegian User Support and Operations Centre) in Norway.

4. The OVT was carried out in June 2015. Specific issues that needed to be tested included the staggered hydration and 4 versus 6 days of growth. Overall, the operations were successful.

5. The PI team has participated in several teleconferences with NASA Ames as well as N-USOC during the OVT and to discuss the experimental procedures/requirements.

6. The PI attended the American Society for Plant Biology (ASPB) meeting in July 2015 and presented a poster on the first flight experiment (Plant Signaling) and the PI team will attend the American Society for Gravitational and Space Research (ASGSR) meeting in November 2015.