NOTE: End date changed to 4/30/2023 per NSSC information (Ed., 5/4/22)

NOTE: End date change to 4/30/2022 per NSSC information (Ed., 4/2/21)

NOTE: End date change to 4/30/2021 per NSSC information (Ed., 3/10/2020)

NOTE: End date changed to 4/30/2020 per PI (Ed., 3/4/19)

NOTE: End date changed to 4/30/2019 per NSSC information (Ed., 8/16/18)

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis; C allocation; water use efficiency (WUE); vascular plant growth/development; vasculature performance; auxin transport; and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi “omics” effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome “discrepancies” result from over-simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

Presentation to Science Mission Directorate: Lewis, NG. "Multi-omics analyses of lignin reduced Arabidopsis lines on ISS." NASA’s Associate Administrator of the Science Mission Directorate (SMD), Washington DC, December 2, 2021. This presentation described several of the exciting new insights gained from the ISS studies.

The overall study had 6 Arabidopsis lines, one wild type (WT), a transgenic line expressing a protein involved in the carbon capture mechanism (CCM), as well as four lines of lignin-reduced arogenate dehydratase mutants (adt), with these encompassing a single mutant (adt5), a double mutant (adt4/5), a quadruple mutant (adt3/4/5/6), and the adt3/4/5/6 mutant expressing CCM.

Photosynthesis measurements on the ISS (and also KSC) deployed portable handheld pulse-amplitude modulated (PAM) devices, these being the first PAM photosynthesis measurements in low Earth orbit (LEO). [PAM measurements estimate the efficiency with which photosystem II (PSII) utilizes light energy for individual plants.]

Relative growth and health of the various Arabidopsis lines in the APH (on either the ISS or at KSC) were monitored through assessment of daily changes in rosette size area and plant color, this being recorded/measured telemetrically. These showed a consistent pattern of slower rosette growth on the ISS relative to KSC Ground Controls. Flowering stem growth estimates and their abilities to have an upright (vertical/near vertical) orientation were also assessed. As flowering progressed, most main flowering stems and subsidiary stems on the ISS in both APH Grow-outs deviated unusually from vertical growth.

Lignin levels were quite similar for KSC Ground Control WT and CCM lines, whereas adt mutants showed increasingly lower lignin contents depending on the mutant. Although a similar trend was noted in ISS plant lines, their levels were further reduced due to plant growth/development/lignification delays. However, reduced lignin levels did not result in vertical stem orientation, suggesting that such levels are not compensated for in microgravity on the ISS. Anatomical analysis of available ISS stem cross-sections indicated some vasculature abnormalities, as compared to the KSC Ground Controls.

Proteomics and Transcriptomics data were obtained for both Grow-outs, together with metabolomics (including lipidomics) analyses). Meaningful integration of multi-omics data is a well-known challenge across all biology disciplines. Accordingly, our inter-disciplinary team effort is largely now focusing on integration and analysis of these data. Our approach is designed to tease out key ISS spaceflight environment effects relative to KSC Ground Control counterparts. Significant progress has been – and continues to be – made to integrate/compare the diverse multi-omics datasets to gain both new and comprehensive insights into the systems-level response(s) to the ISS spaceflight microgravity environment with the Arabidopsis genotypes. For example, pathway analysis was accomplished using targeted and untargeted approaches. The targeted approach involved extracting features from each dataset known to be associated with various structural (biochemical) pathways, and by comparing fold changes in ISS vs KSC Ground Controls across genotypes. The untargeted method involved the use of a LANL developed tool OPaver (Omics Pathway Viewer). OPaver enables us to map differential expression/abundance values onto curated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps for transcriptomics, proteomics, and metabolomics datasets. Preliminary analysis indicates that some metabolic pathways were augmented by ISS spaceflight conditions vs KSC Ground Controls and are further being evaluated. Additionally, we conducted a targeted analysis of genes/proteins involved in chorismate/shikimate, flavonoid, and phenylpropanoid pathways. In general, we observed a global decrease in genes/proteins involved in the phenylpropanoid pathway for plants grown on ISS compared to KSC Ground Controls; this correlates with the observed decrease in measured lignin contents grown on ISS compared to KSC, corroborating the overall decrease in abundance of structurally related features across datasets in ISS samples.

NOTE: End date change to 4/30/2022 per NSSC information (Ed., 4/2/21)

NOTE: End date change to 4/30/2021 per NSSC information (Ed., 3/10/2020)

NOTE: End date changed to 4/30/2020 per PI (Ed., 3/4/19)

NOTE: End date changed to 4/30/2019 per NSSC information (Ed., 8/16/18)

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis; C allocation; water use efficiency (WUE); vascular plant growth/development; vasculature performance; auxin transport; and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi “omics” effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome “discrepancies” result from over-simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

Oral Presentations:

"An integrated omics guided approach to lignification and gravitational responses: Current progress and the ISS APH prototype (PH-01)." NASA Lunch Time Meeting, November 24, 2020 (Virtual Seminar).

1. ISS and KSC Grow-outs

• The first grow-out in the APH (APH-01) was initiated by NASA astronaut Ricky Arnold on ISS on June 8, 2018 (Day 0). On June 22, 2018 (Day 14), manual FluorPen photosynthesis measurements were carried out, followed by Arabidopsis seedlings thinning to give one viable plant per growth site in the APH. “Thinnings,” to remove “excess” numbers of seedlings, were placed in foil, immediately frozen, and stored in the GLACIER freezer at –160 °C on ISS.

• Spaceflight environment effects were observed with the remaining plant lines growing in APH-01. Following the thinning operation, several plants unexpectedly died, and others grew somewhat smaller in size. The impact on our experiment was that only one harvest at 45 days was possible in order to ensure sufficient plant material availability for our multi-omics analysis. FluorPen photosynthesis measurements were, however, again conducted after 31 days (4 weeks and 3 days) of growth (July 9, 2018). All remaining plants were finally harvested on ISS on July 23, 2018, and immediately frozen and stored in the GLACIER freezer at –160 °C (as for the thinnings). The frozen ISS-grown plant specimens were returned to Earth on January 13, 2019, and transported/delivered frozen to the Institute of Biological Chemistry (IBC), Washington State University (WSU), on January 15, 2019.

• The corresponding Ground control APH-01 study, at Kennedy Space Center (KSC), was initiated a week later (June 15, 2018; Day 0), with FluorPen photosynthesis measurements/thinnings done on June 29, 2018 (Day 14). FluorPen photosynthesis measurements were again carried out on July 13, 2018 (Day 28), with final harvest on July 27, 2018 (Day 42). Ground control plants were transported to Institute of Biological Chemistry (IBC), Washington State University (WSU) on January 25, 2019.

• The second ISS grow-out was initiated by astronaut Serena Auñón-Chancellor on September 18, 2018. Following FluorPen photosynthesis measurements, the subsequent thinning operation on ISS (October 2, 2018) removed the “excess” plants that were immediately frozen and stored in the GLACIER freezer at –160 °C. The thinning procedure may have adversely affected growth/development of some Arabidopsis plants in APH-01 with several again dying after thinnings had been harvested. FluorPen measurements were carried out on ISS on October 19, 2018 (Day 31). Slightly slower Arabidopsis growth was observed in the ISS APH facility, relative to our Ground control expectations, with only a single harvest at ~6 weeks (November 2, 2018; Day 45) being possible, in order to obtain the required plant material for our multi-omics analysis. The 6 week old harvested plants were immediately frozen and stored in the GLACIER freezer at –160 °C. All frozen specimens were returned to Earth on January 7, 2020, and transported/delivered frozen to IBC/WSU on January 09, 2020.

• The corresponding Ground control was initiated at KSC a week later (September 25, 2018; Day 0), with FluorPen photosynthesis measurements/thinning occurring on October 9, 2018 (Day 14), FluorPen photosynthesis measurements were again conducted on October 26, 2018 (Day 31), with the final harvest being on November 9, 2018 (Day 45). The Ground control plants were transported/delivered to IBC/WSU on January 09, 2020.

• Covid-19 Impacts: The Progress Report herein for this reporting period focuses upon the continued analyses of Arabidopsis plants on ISS and KSC (Ground controls) from both Grow-out #1 and Grow-out #2. Progress was, however, significantly delayed due to Covid-19. Disruptions included personnel being unable to work for extended periods of time, high-end instrumentation required for our multi-omics analyses being inaccessible due to restrictions on working, and manufacturer delays in providing materials and supplies.

2. Consortium Member David Hanson (University of New Mexico, UNM) – Morphology

Estimated colorimetric lignified vasculature distribution analyses in inflorescence “stem” cross-sections using both staining and microscopy were completed. Samples were processed by standard microscopy sectioning protocols with Toluidine Blue (TBO) staining, which stains the lignified vasculature blue and other non-lignified tissue pinkish red. These different staining patterns were used to estimate the ratio of lignified vasculature to non-lignified tissue in images.

Grow-out #2

The tendency of growing stems in the ADT genotypes to turn toward the Science Carrier base was similar to Grow-out #1. ADT knockout mutants with less stained vasculature had stems touching the base of the Science Carrier faster than WT or WT-CCM lines. Stem cross-sectional area was also variable between both Ground and ISS samples, but both showed expected patterns of colorimetric lignified vasculature to non-lignified tissue ratio with ADT disruptions. There was a lower colorimetric lignified vasculature to non-lignified tissue ratio in knockout mutants (lowest in the quadruple knockouts) and more in WT lines. Lignin modified ADT knockout mutants grown aboard ISS again showed non-uniform distribution of stained lignified vasculature around the lignified vasculature.

3. Consortium Members Laurence Davin and Norman Lewis (WSU/IBC) – Arabidopsis Multi-omics Sample Preparation

ISS and KSC (Ground control) samples from Grow-out #2 were processed at WSU/IBC as described below in order to obtain: • RNAs for transcriptomics, • Proteins for proteomics, • Lipids for lipidomics, • Metabolites for metabolomics, • Intact stem tissues for microscopy analysis, • Ground stem tissue for lignin and stable isotope analyses.

Samples from both ISS and Ground controls (KSC), stored individually in double-pocketed foil bags (one pocket containing rosette leaves, and the other pocket containing stems with flowers, siliques, and cauline leaves), were each removed from the –80 degrees C freezer and placed in dry ice. Each sample was first weighed in its foil bag, and then placed into liquid nitrogen. Following sample removal for processing, each foil bag was re-weighed in order to obtain the fresh (frozen) weight of each sample.

Arabidopsis rosette leaf tissue sample DNA screening: Individual Arabidopsis ADT mutant and WT and WT-CCM lines in their respective positions in the APH Science Carrier were analyzed using a REDExtract-N-Amp ™ Plant PCR Kit (SIGMA) protocol to confirm each genotype was in the assigned position.

4. Arabidopsis Multi-omics Analyses

In addition to the ongoing analysis of Grow-out #1 plant lines (ISS and Ground controls), these were also initiated on specimens from ISS Grow-out #2 tissues, and the corresponding KSC (Ground control) samples. Following sample preparation as indicated above, these were sent to Los Alamos National Lab (LANL), Pacific Northwest National Lab (PNNL), and UNM (University of New Mexico) consortium members, respectively.

4.1. Consortium Member Shawn Starkenburg (LANL) – Transcriptomics

Grow Out #1

An analysis pipeline was additionally constructed to evaluate possible single nucleotide polymorphism (SNP) level variation in Ground vs. ISS samples to test the impact of microgravity/ISS spaceflight environment on the mutation rate. Fewer mutations were found in stem than leaf samples, regardless of ISS or Ground conditions. Additional validation/analysis is underway to confirm these results.

Grow Out #2

Upon receipt of the total RNA from leaves and stems (September 2020), sequencing libraries (n=94) were constructed for the second set of ISS and Ground control samples. Prior to sequencing all transcriptome samples, a test sample set using 5 random Arabidopsis thaliana transcriptome libraries (5 million reads) were sequenced to verify sample integrity. Sequencing of this test sample set resulted in a predicted pattern of base calls throughout the length of the read when mixed with other types of DNA libraries (non-Arabidopsis samples), indicating that viable transcriptomes can be generated from the samples from the Grow-out #2 ISS experiment.

To maximize sequence integrity for additional RNA mutation analysis of the 2nd ISS experiment, a sequencing approach was designed to minimize the impact of sequencing errors and batch effects (run-to-run variability). In short, each sample is to be sequenced a minimum of three times, combining the reads acquired across several sequencing runs until the required sequencing depth is achieved (approximately 25 million reads per sample). Our first batch of samples is being currently sequenced and the plan is to complete sequencing by the end of March 2021.

Covid-19 Impact

Covid-induced shutdowns of Los Alamos National Laboratory (LANL) facilities in the Spring of 2020 prohibited timely processing and transfer of total RNA preps from WSU to LANL. Additionally, domestic travel by LANL personnel to collect samples from WSU was not permitted until late September 2020, despite the fact that the RNA was ready for collection in the Spring.

LANL continues to operate under reduced capacity (approximately 75% efficiency) and reagent & materials sourcing delays for processing of samples continues to hamper research progress. However, it is anticipated that LANL facilities will be fully operational in the summer of 2021.

4.2. Consortium Members Laurence Davin and Norman Lewis (WSU/IBC) – Arabidopsis Metabolomics

Grow-out #2

ISS and KSC (Ground control) stem and leaf samples from Arabidopsis Grow-out #2 were individually processed, extracted, and then subjected to metabolomics analyses. Specifically, each ground leaf and stem sample (~150 to 200 mg) from four biological replicates of six Arabidopsis lines that were grown in APH units on ISS and at KSC (Ground control), respectively, were individually extracted with aqueous methanol. The metabolite extracts of each leaf and stem sample, after solvent extraction, were next individually subjected to UPLC-qTOF-MS for metabolomics analyses. LC-MS analysis and identification of metabolites in each sample were performed as previously described.

In comparison to Grow-out #1, once again the metabolites analyzed in Grow-out #2 were mainly annotated as apocarotenoids, flavonoids, glucosinolates, galactolipids, phenolic glucosides, and phenylpropanoids, respectively. Each metabolite in these classes was identified/annotated and quantified in terms of naringenin equivalents (internal standard).

Next, as one example, statistical analyses were performed to compare the metabolomic profiles of Arabidopsis WT and adt3/4/5/6 lines, grown on ISS and at KSC (Ground control) in their respective APH units. This involved pair-wise comparisons of the metabolites obtained for ISS and KSC (Ground control) samples, after normalization of data to the internal standard naringenin in XCMS.

Processing the metabolomics data resulted in clear segregation of ISS and KSC (Ground control) Arabidopsis WT and adt3/4/5/6 lines in terms of either leaf or stem metabolite clusters; indeed, all sample sets were readily distinguished as to both genotype and whether they were ISS or Ground control (KSC) grown (data not shown).

As further examples, heatmap diagrams were also generated from the metabolomics data for WT Ground control (KSC), WT (ISS), adt3/4/5/6 Ground control (KSC) and adt3/4/5/6 (ISS) samples. It was evident from these heatmaps that the average relative metabolite levels of leaf and stem samples for WT Ground control (KSC) and adt3/4/5/6 Ground control (KSC) slightly differed in their levels compared to WT (ISS) and adt3/4/5/6 (ISS).

ISS and KSC (Ground control) stem samples from Arabidopsis Grow-out #2, processed and extracted exactly as for Grow-out #1, are in progress for lignin content/composition analyses.

4.3. Consortium Members (Kim Hixson and Mary Lipton, PNNL) – Proteomics and Lipidomics

Grow-out #1:

Proteomics: Analysis of Grow-out #1 proteins were further overlaid onto KEGG pathway maps to visualize relevant specific proteome changes. The auxin pathway was also explored as auxin signaling is associated with gravitropism. It was observed that the proteome response was much greater in stem tissue as compared to leaves. In the more extreme mutants (i.e., adt4/5, adt3/4/5/6), the increases or decreases in specific proteins showed converse abundance profiles. That is, auxin associated proteins highly increased in leaf tissue were conversely lower in abundance on ISS compared to Ground control proteins in the stem tissues.

We also examined the log2 ratio z-scores [ISS grown/Ground control] onto the immediate upstream (shikimate/chorismate) and downstream (phenylpropanoid) pathways relative to arogenate dehydratase. In almost all enzymes detected in both tissues, and in different ADT knock-out combinations, we observed these enzymes to be increased in abundance. It seems that the ISS spaceflight environment has a consistent and significant effect of increasing abundance of these enzymes. A notable exception was observed in the first enzyme of this pathway in the leaves of adt5, namely 3-deoxy-7-phosphoheptulonate synthase. Interestingly, there was a significant decrease in abundance observed in the ISS grown leaves compared to those grown as Ground control.

In these analyses, there was a mix of enzymes that were increased as well as decreased in abundance. The greatest increases or decreases in abundance were identified in the double knock-out (KO) ADT mutant adt4/5 both in leaves and stems. The KO mutant with more ADTs knocked out, adt3/4/5/6 showed more moderate abundance changes. In both stems and leaves and in all examined ADT KOs, trans-cinnamate 4-hydroxylase was increased in the ISS grown plants as compared to the Ground control plants. This variety of increases and decreases in a single pathway with plants that contain various ADT isoenzymes provisionally demonstrates how the phenylpropanoid pathway enzymes are collectively controlled by additional mechanisms under ISS spaceflight environment conditions beyond just the amount of phenylalanine available to the pathway.

Lipidomics: Analyses on pertinent mass spectrometer platforms and pipelines have all been completed for WT, ADT mutant, and CCM-engineered Arabidopsis from Grow-out #1. The data is currently being assessed and interpreted.

Grow-out #2

Proteomics: Precipitated proteins were extracted in an identical way by WSU IBC personnel as for the Grow-out #1 experiments and provided to Pacific Northwest National Laboratory (PNNL). At PNNL, precipitated proteins were then individually solubilized in a denaturing solution and digested into peptides using trypsin, with each sample desalted and quantified. As in the Grow-out #1 experiment, the isobaric tagging protocol (iTRAQ) method was used to label peptides and to determine relative quantitation of each peptide in each sample. We are currently waiting for all of the proteomics data from the Grow-out #2.

Lipidomics: Lipid samples for Grow-out #2 have also been completed and the two grow-out datasets are currently being integrated and analyzed for assessment of the significant lipid changes in leaves and stems due to plants being subjected to ISS spaceflight environment growth conditions.

Covid-19 Impact

WSU/IBC provided samples of all stem and leaf tissues from Grow-out #2 from ISS and the corresponding Ground controls. Covid-19 stay-at-home orders and instrumental resource diversion at the Environmental Molecular Sciences Laboratory (EMSL) at the Pacific Northwest National Laboratory (PNNL), significantly delayed progress on the second flight proteomics analysis, until late Fall 2020.

5. Consortium Member David Hanson (UNM) – 13C Analyses

13C analyses were carried out on plant leaf samples from Grow-out #1 (Ground control and ISS) and will be completed on plant leaf samples from Grow-out #2 (Ground control and ISS). Samples have been freeze-dried and will be packed (2 mg) for analysis via isotope ratio mass spectroscopy through the Center for Stable Isotopes Facility at UNM. 13C/12C analyzes will provide insight into the photosynthetic function and water use efficiency (WUE) over the lifetime of the plants during their growths in the APH chambers. This is important in understanding stomatal usage and closure that may help detect symptoms of stress from flight.

Covid-19 Impact

Delay on these analyses has occurred because of back order issues on the supplies needed due to COVID-19.

NOTE: End date change to 4/30/2021 per NSSC information (Ed., 3/10/2020)

NOTE: End date changed to 4/30/2020 per PI (Ed., 3/4/19)

NOTE: End date changed to 4/30/2019 per NSSC information (Ed., 8/16/18)

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis; C allocation; water use efficiency (WUE); vascular plant growth/development; vasculature performance; auxin transport; and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi 'omics' effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome 'discrepancies' result from over-simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

2). We have partnered with Ms. Kathy Lucchesi (K-7/8 teacher), at McCaffrey Middle School in Galt, California, and their largely Hispanic students. Supplemental funding was provided to the school by NASA and the California Space Grant Consortium so that these middle school students can safely follow and utilize many of the plant growth and development protocols developed for the International Space Station (ISS) experiments. One purpose here is that the students grow plants under similar conditions and obtain information and insights on how the research impacts or benefits life on Earth and beyond (in the future).

Written materials on, and seeds for, the experiments at hand are also routinely provided. The additional aim here is in helping teach and inspire these young students about the joys and fun of the scientific method in experimental plant biology. Periodically, the middle school students present results to Dr. Lewis over where such work is routinely evaluated.

3). The Lewis lab hosted Pullman Lego Robotics Team to discuss their project and help them prepare for their competition (January 2019).

4). David Hanson was the lead organizer for a NASA supported In-flight Education Downlink with Astronaut Christina Koch on the International Space Station ( https://news.unm.edu/news/nasa-astronaut-chats-live-with-500-students-at-unm ). This event had an audience of over 700 people, including over 500 K-12 students that traveled to University of New Mexico (UNM) to participate in person as well as others watching via an internet live stream (live.unm.edu). The event had many hands-on activity booths from local STEM (Science, Technology, Engineering, and Mathematics) education programs and guest presentations by NASA scientists and student interns. It was also co-hosted by The Children’s Hour radio program and part of a subsequent hour-long show that was broadcast nationwide. Research from APH-01 was highlighted in the live event (found online at live.unm.edu as well as directly through the NASA past downlinks site- https://www.nasa.gov/audience/foreducators/stem-on-station/downlinks.html ) and on the radio show ( https://www.childrenshour.org/kids-in-space/ ).

5). David Hanson gave two lectures, one for the Science on Tap series and the other for the Interesting Conversations Series (see Bibs Patents Awards).

6). Miss Bianca Serda, a Hispanic student with Dr. Hanson gave three oral and three poster presentations. She was the recipient of a NASA Space Life Science Training Program (SLSTP) fellowship at Ames Research Center (from June 10, 2019 to August 16, 2019), results of which she presented at the American Society for Gravitational & Space Research (ASGSR) meeting in Denver.

Oral Presentations:

• Serda, B., Turpin, M., Hudson, P., and Hanson, D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” UNM Center for Stable Isotopes Seminar, Albuquerque, NM, October 2019

• Serda, B., Turpin, M., Lewis, N., and Hanson, D. “Plants in Space: Do Lignification Levels and Microgravity Interact to Impact Photosynthesis? University of New Mexico Department of Biology 28th Annual Research Day, Albuquerque, NM, March 2019

Poster Presentations:

• Serda, B., McKaig, J., Waters, S., Venkateswaran, K.J., Smith, D.J. Methylation pattern detection of the genome of Bacillus pumilus strain SAFR-032. 35th Annual Meeting American Society for Gravitational and Space Research, Denver, CO, November 20 - 23, 2019

• Serda, B., Turpin, M., Hudson, P., and Hanson, D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” SACNAS Conference, Honolulu, HI, November 2019

• Serda, B., Turpin, M., Hudson, P., and Hanson, D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” New Mexico AMP Conference, New Mexico State University, Albuquerque, NM, October 2019.

7). This project has been showcased on the Space Biology Facebook page at: https://www.facebook.com/spacebiology/posts/

8). Dr. Lewis and Dr. Davin were judges for the High School Student Poster competition at the annual ASGSR meeting in Denver, CO, November 20 – 23 2019.

1. ISS and KSC grow-outs:

• The first grow-out in the APH (APH-01) was initiated by NASA astronaut Ricky Arnold on ISS on June 8, 2018 (Day 0). On June 22, 2018 (Day 14), manual FluorPen photosynthesis measurements were carried out, followed by thinning of the Arabidopsis seedlings to give one viable plant per growth site in the APH. The “thinnings,” to remove “excess” numbers of seedlings, were placed in foil, immediately frozen, and stored in the GLACIER freezer at –160°C on ISS.

• Spaceflight environment effects were observed with the remaining plant lines growing in APH-01. Following the thinning operation, several plants unexpectedly died, and others grew somewhat smaller in size. The impact on our experiment was that only one harvest at 45 days was possible in order to ensure sufficient plant material availability for our multi-omics analysis. FluorPen photosynthesis measurements were, however, again conducted after 31 days (4 weeks and 3 days) of growth (July 9, 2018). All remaining plants were finally harvested on ISS on July 23, 2018, and immediately frozen and stored in the GLACIER freezer at –160°C (as for the thinnings). The frozen ISS-grown plant specimens were returned to Earth on January 13, 2019, and transported/delivered frozen to the Institute of Biological Chemistry (IBC), Washington State University (WSU), on January 15, 2019.

• The corresponding ground control APH-01 study, at Kennedy Space Center (KSC), was initiated a week later (June 15, 2018; Day 0), with FluorPen photosynthesis measurements/thinnings done on June 29, 2018 (Day 14). FluorPen photosynthesis measurements were again carried out on July 13, 2018 (Day 28), with the final harvest on July 27, 2018 (Day 42). Ground control plants were transported/ delivered to IBC/WSU on January 25, 2019.

• The second ISS grow-out was initiated by astronaut Serena Auñón-Chancellor on September 18, 2018. Following FluorPen photosynthesis measurements, the subsequent thinning operation on ISS (October 2, 2018) removed the “excess” plants that were immediately frozen and stored in the GLACIER freezer at –160°C. The thinning procedure may have adversely affected growth/development of some Arabidopsis plants in APH-01 with several again dying after the thinnings had been harvested. FluorPen measurements were carried out on ISS on October 19, 2018 (Day 31). Slightly slower Arabidopsis growth was observed in the ISS APH facility, relative to our ground control expectations, with only a single harvest at ~6 weeks (November 2, 2018; Day 45) being possible, in order to obtain the required plant material for our multi-omics analysis. The 6 week old harvested plants were immediately frozen and stored in the GLACIER freezer at –160°C. All frozen specimens were returned to Earth on January 7, 2020, and transported/delivered frozen to IBC/WSU on January 09, 2020.

• The corresponding ground control was initiated at KSC a week later (September 25, 2018; Day 0), with FluorPen photosynthesis measurements/thinning occurring on October 9, 2018 (Day 14), FluorPen photosynthesis measurements were again conducted on October 26, 2018 (Day 31), with the final harvest being on November 9, 2018 (Day 45). The ground control plants were transported/delivered to IBC/WSU on January 25, 2019.

• This Progress Report largely focuses on analyses of Arabidopsis plants on ISS and KSC (ground controls) from Grow-out #1, with Grow-out #2 data as available.

2. Consortium Member (David Hanson Lab, University of New Mexico, UNM) Activities for Arabidopsis Grow-out #1 and Grow-out #2 on APH-01 (ISS and KSC)

2.1. Estimated growth behavior of Arabidopsis wild type (WT) and arogenate dehydratase (adt), carbon capture mechanism (CCM), and adt/CCM mutant plants on ISS and KSC (ground controls)

Photographic images of all six Arabidopsis lines (described above), during growth and development, were downloaded to Earth twice a day, with the images being processed to estimate relative growth of rosette tissue size for each plant/plant line. In both grow-outs, growth/development lagged on ISS relative to KSC ground contols. The same trends were observed in both grow-outs. In addition, estimates of flowering stem growth and their abilities to have an upright (vertical) orientation were made for both ISS and ground control (KSC) plant lines. In all ISS-grown plants, an off-vertical growth displacement of the stems was observed regardless of the plant line being analyzed. This was in contrast to the KSC grown lines that generally had vertically growing flowering stems.

2.2. Photosynthesis measurements

Fluorpen-based estimations of photosynthetic function (chlorophyll fluorescence) showed much greater variability and overall higher values on ISS relative to ground contols. These data consisted of light-adapted measurements of pulse amplitude modulated (PAM) chlorophyll fluorescence at lower (150 µmol photons m–2 s–1) and higher (800 µmol photons m–2 s–1) light levels, these being collected at least 20 minutes after opening the APH to allow for effects from the high ISS cabin CO2 (4000 ppm versus 400 ppm in the APH) to equilibrate. Two light intensities were used to determine the electron transport rate (ETR) of photosynthesis in growth conditions and the ability to be stimulated by higher light that was near saturation in pre-grow-out experiment trials on Earth.

3. Consortium Members (Laurence Davin and Norman Lewis, WSU/IBC) - Arabidopsis Multi-omics Sample Preparation

ISS and KSC (ground control) rosette leaf and stem tissues samples were processed individually at WSU/IBC as described below in order to obtain: • RNAs for transcriptomics, • Proteins for proteomics, • Metabolites for metabolomics, • Intact stem tissues for microscopy analysis, • Ground stem tissue for lignin and stable isotope analyses.

Samples from both ISS and ground controls (KSC), stored individually in double-pocketed foil bags (one pocket containing rosette leaves, and the other pocket containing stems with flowers, siliques, and cauline leaves), were each removed from the –80ºC freezer and placed in dry ice. Each sample was first weighed in its foil bag, and then placed into liquid nitrogen. Following sample removal for processing, each foil bag was re-weighed in order to obtain the fresh (frozen) weight of each sample.

4. Arabidopsis multi-omics analyses

These have been initiated on specimens from ISS Grow-out #1 tissues, and the corresponding KSC (ground control) samples. Following sample preparation as indicated above, these were sent to Los Alamos National Lab (LANL), Pacific Northwest National Lab (PNNL), and UNM consortium members. The data will be re-processed in future work in order to compare and integrate all multi-omics analysis results per individual plant.

4.1. Consortium member (Shawn Starkenburg, LANL) - Transcriptomics

All samples provided by WSU/IBC, and meeting Quality Control expectations, from Arabidopsis Grow-out #1 were provided to LANL for analysis. These were sequenced using the LANL Illumina sequencer at an approximate depth of 100 fold coverage. Total number of reads for each sample ranged from 10,000 to 50,000. Each tissue, leaves and stems, were sequenced independently. Raw reads were trimmed for quality using FaQCs and mapped to the Arabidopsis thaliana genome using HiSat2. Reads were counted using FeatureCounts and analyzed for differential expression using edgeR, implemented in R.

Differential gene expression analysis was preliminarily performed in two ways: examination of i) global gene expression differences and ii) specific pathways differentially regulated, based on previous work with adt lignin deficient mutants. Multidimensional clustering of samples demonstrated that samples clustered primarily by tissue type, then by ISS/KSC origin.

For global gene expression analysis, ISS Arabidopsis genotypes were compared to respective tissue WT (KSC) ground control samples. A total of 1,403 genes were differentially expressed, as compared to the WT KSC ground control data. In this preliminary analysis, most genes were differentially expressed in leaves, as compared to stems. Additionally, ISS samples, as compared to the KSC (ground control) counterparts, displayed most differentially expressed genes. A preliminarily examination of the potential function of genes that were significantly differentially regulated in ISS leaf samples was carried out.

Specific analyses were performed to examine effects upon phenylpropanoid pathways in lignin-reduced mutants. Significant upregulation of phenylpropanoid pathway genes amongst mutant genotypes was observed relative to WT, but also for WT samples grown in ISS, provisionally suggesting that pathways examined were biologically relevant.

4.2. Consortium members (Laurence Davin and Norman Lewis, WSU/IBC) – Arabidopsis lignin analyses and metabolomics

ISS and KSC (ground control) samples from Arabidopsis Grow-out #1, processed as described at the beginning of Section 3, were subjected to lignin content/composition and metabolomics analyses, for each stem and leaf tissue type.

4.2.1. Arabidopsis estimated lignin analyses

The overall average lignin levels were estimated (in micromoles of thioacidolysis products per gram cell wall residue (CWR)) for the Arabidopsis stems from the six different Arabidopsis lines grown on ISS and at KSC, respectively, in their corresponding APH units. With the exception of the adt5 mutant, most lines (average of 3-4 plants/line) gave similar lignin levels (contents/compositions) for both ISS and KSC (ground control) APH-grown plants, whether the line was reduced in lignin amount via ADT mutations or not.

This data was further scrutinized, whereby each individual plant lignin level for all six lines was determined. This clearly established that most plant lines had fairly similar lignin levels (or similar variability) for ISS and KSC plant lines grown in the 2 APH units, with the exception of three of the four ISS grown adt5 mutants; this anomaly presumably resulted from 3 of their 4 growth sites in the APH on ISS not receiving adequate watering. Taken together, the data would suggest that constitutive lignification levels in all six Arabidopsis lines were not greatly affected when grown on ISS, relative to the ground controls at KSC; similar observations were made by us on growing dwarf wheat on the Space Shuttle.

4.2.2. Arabidopsis metabolomics

Metabolomics analyses were conducted on each Arabidopsis plant stem and leaf sample from the six Arabidopsis lines that were ISS and KSC (ground control) grown in their respective APH units, as follows: Each sample analysis involved UPLC-qTOF-MS for metabolomics. LC-MS analysis and identification of metabolites in each sample were performed as previously described.

Representative chromatograms of the main Arabidopsis metabolite classes present in leaf and stem tissues of the WT line, grown in APH units on ISS and KSC, respectively, were obtained. Under these chromatographic conditions, the metabolites analyzed were mainly annotated as apocarotenoids (A), flavonoids (F), glucosinolates (G), galactolipids (GL); phenolic glucosides (PG), and phenylpropanoids (P), respectively.

It was of considerable interest to additionally determine whether there were significant changes in the plant metabolic profiles grown on ISS, relative to the ground controls at KSC. An example shown is that of the comparison of the metabolic profiles of Arabidopsis WT and adt3/4/5/6 lines, grown on ISS and at KSC (ground control) in their respective APH units. This involved pair-wise comparisons of the metabolites obtained for ISS and KSC (ground control) samples, after normalization of data to the internal standard naringenin in XCMS. The respective sPLS-DA (Sparse Partial Least Squares Discriminant Analysis) plots for the Arabidopsis leaf and stem samples (4 biological and 2 technical replicates each) for WT ground control (KSC), WT ISS, adt3/4/5/6 ground control (KSC) and adt3/4/5/6 ISS) samples were generated by performing statistical analyses of identified secondary metabolites using MetaboAnalyst version 4.0. Processing the metabolomics data in this way resulted in clear segregation of ISS and KSC (ground control) WT Arabidopsis leaf metabolite clusters, and also those of the adt3/4/5/6 lines from the ISS and KSC samples. While there was some very minimum overlap for each of these clusters, all sample sets were readily distinguished as to both genotype and whether they were ISS or ground control (KSC) grown. An analogous situation holds for the stem samples.

4.3. Consortium members (Kim Hixson and Mary Lipton, PNNL) – Proteomics, phosphoproteomics, and lipidomics

4.3.1. Proteomics

WSU/IBC provided samples of all stem and leaf tissues for individual Arabidopsis plants prepared for global proteomics and phospho-proteomics analyses at PNNL. Samples received were of precipitated proteins. Precipitated proteins were individually solubilized in a denaturing solution and digested into peptides using trypsin. The isobaric tagging protocol (iTRAQ) method was used to quantitatively determine the abundances of peptides in each sample, with each peptide identified and quantified by searching the fragmentation pattern against the Arabidopsis genome.

For the global proteomics in Arabidopsis Grow-out #1, 16,499 proteins could be identified by matching against the Arabidopsis genome, this corresponding to almost 60% of the protein coding regions. A PLS-DA showed separations of leaf and stem samples from the KSC (ground control) and ISS in Arabidopsis Grow-out #1. This PLS-DA plot, along with coefficient of variation analysis between ISS grown plants and KSC ground control plants, also showed that ISS-grown Arabidopsis leaf and stem samples were more variable than the KSC (ground control) plants.

The proteomics data provisionally suggest that plants grown on ISS had much stronger responses in their proteomes, as compared to plants grown at KSC (ground control). Several more proteins increased in abundance as ADT and lignin levels were reduced. These results indicate that as lignin is reduced, and plants are grown on ISS, they perhaps perceive more stress in the form of radiation and microbial perception/susceptibility, the result of which triggers higher numbers of cell signaling, transport, and exosome related proteins.

Additional work is underway to complete the proteomic analyses of each leaf and stem sample from all six Arabidopsis lines in Grow-out #1.

4.3.2. Phosphoproteomics and lipidomics

Analyses on pertinent mass spectrometer platforms and pipelines have all been completed for WT, adt mutant and CCM-engineered Arabidopsis from Grow-out #1. The data is currently being assessed and interpreted.

5. Consortium member (David Hanson Lab, UNM) 13C analyses

The 13C analyses were carried out on plant leaf samples (ISS and KSC grown) that were ground to a powder and provided by the WSU/IBC in May 2019. The 13C data obtained to date, however, from the six Arabidopsis Grow-out #1 samples showed few differences between genotypes grown on ISS vs KSC ground controls, although ISS plant tissues gave much higher variability in these measurements.

NOTE: End date changed to 4/30/2020 per PI (Ed., 3/4/19)

NOTE: End date changed to 4/30/2019 per NSSC information (Ed., 8/16/18)

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis; C allocation; water use efficiency (WUE); vascular plant growth/development; vasculature performance; auxin transport; and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi 'omics' effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome 'discrepancies' result from over-simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

2). We have partnered with Ms. Kathy Lucchesi (K-7/8 teacher), at McCaffrey Middle School in Galt, California, and their largely Hispanic students. Supplemental funding was provided to the school by NASA and the California Space Grant Consortium so that these middle school students can safely follow and utilize many of the plant growth and development protocols developed for the International Space Station (ISS) experiments. One purpose here is that the students grow plants under similar conditions and obtain information and insights on how the research impacts or benefits life on Earth and beyond (in the future).

Written materials on, and seeds for, the experiments at hand are also routinely provided. The additional aim here is in helping teach and inspire these young students about the joys and fun of the scientific method in experimental plant biology. Periodically, the middle school students present results to Dr. Lewis over where such work is routinely evaluated.

Astronaut Sunita Williams, Dr. Lewis (Washington State University), and Dr. Sato (NASA Ames) visited the school on April 13, 2018. Astronaut Williams gave presentations on her time on International Space Station and answered questions from students. The Galt Herald ( http://www.galtheraldonline.com/news/mccaffrey-students-visited-by-space-explorer/article_144aa81a-4334-11e8-842f-f7106ee5d2d7.html ) and the Lodi News-Sentinel ( https://www.lodinews.com/news/article_982e5434-3faa-11e8-b4e1-db589601c0df.html?mode=story ) wrote articles on her visit, and the visit was further publicized through the NASA Space Biology Facebook ( https://www.facebook.com/spacebiology/ ).

Later, McCaffrey Middle School Students presented their work on #PlantHabitat01 to the Galt School Board on (See post at: https://www.facebook.com/spacebiology/posts/917222148451703 ).

Ms. Lucchesi, her students, and Dr. Lewis visited NASA AMES Research Center, May 28, 2018. The students presented their research to NASA scientists, engineers, and managers. The students also handled small satellites, checked out our bone and signaling lab, learned about Astrobiology and Air Revitalization on ISS. (See post at: https://www.facebook.com/spacebiology/posts/931694277004490 ).

Ms. Lucchesi and one of her students traveled to Washington State University, Pullman to visit Dr. Lewis’ and Davin’s laboratory. They had hands-on training from the lab on what is being done with the Arabidopsis plants. (See post at: https://www.facebook.com/spacebiology/posts/1020912004749383 )

The McCaffrey Middle School students were featured on a Good Day Sacramento segment between 8-10 AM, Thursday, October 25, 2018! (See Part 1: https://cbsloc.al/2D4vJMC and Part 2: https://cbsloc.al/2EIfN4d ), with the Lewis lab personnel included from Washington State University (WSU) (via teleconference).

3). On November 7, 2018 and January 3, 2019, the Pullman Droids, a Lego Robotics Team, visited Drs. Lewis and Davin to learn of their work on ISS.

4). Dr. Lewis gave seminars at Tarim University, Alar, Xinjian, China, June 15, 2018, and Southwest University for Nationalities, Chengdu, Sichuan, China, June 24, 2018, entitled “Approach to Lignification on Earth and on the International Space Station.”

5). The New Mexico Space Grant Consortium helped to fund a year-long collaboration between Dr. Hanson and the Explora science museum in Albuquerque. They developed and piloted a monthly course called "Extraterrestrial Botany" for around twenty students across the city who were in 5th through 9th grade. Ten sessions were developed and taught by a team of 2 post docs (1 female), two technicians (both female, 1 Hispanic), a female graduate student, and three undergraduates (2 female, 1 Hispanic), plus Dr. Hanson and two staff members at Explora. The project involved learning about challenges of space exploration, relevance for life on Earth, and the student assisted with data collection and analysis from the APH-01. Feedback from families and Explora was extremely positive and they would like to do more work with us.

6). Miss Bianca Serda, a Hispanic student with Dr. Hanson, gave an oral and 2 poster presentations. She is also the recipient of a NASA Space Life Science Training Program (SLSTP) fellowship at Ames Research Center (from June 10, 2019 to August 16, 2019).

• Serda B, Turpin M, Hudson P, Hanson D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” Presented at the MARC Symposium, Albuquerque, NM, August 2018.

• Serda B, Turpin M, Hudson P, Hanson D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” New Mexico Academy of Science Research Symposium at Albuquerque, NM, October 2018.

• Serda B, Turpin M, Hudson P, Hanson D. “Plants in Space: Interactions between Morphology, Lignification, and Carbon Isotopic Composition.” SACNAS Conference at San Antonio, TX, October 2018.

7). Fans of science in space now can experience fast-moving footage in even higher definition as NASA and ESA (European Space Agency) deliver the first 8K ultra high definition (UHD) video of astronauts living, working and conducting research from the International Space Station. https://www.youtube.com/watch?v=7k2uKb9vCOI . One of the selected studies was our experiment carried out with the Advance Plant Habitat (APH) on ISS.

8) Our experiment has been showcased many many on the Space Biology Facebook page at: https://www.facebook.com/spacebiology/posts/

9). Dr. Lewis and Dr. Davin were judges for the High School Student Poster competition at the annual ASGSR (American Society for Gravitational & Space Research) meeting in Bethesda, MD, October 31 – November 3 2018.

• Carrying out the second Experiment Verification Test (EVT). This was in order to further test conditions that will be employed on International Space Station (ISS).

• Updating the Experiment Requirements Document (ERD) for consideration and approval by the SLPS Program Executive for Space Biology at NASA Headquarters.

• Carrying out two 6-week Arabidopsis grow-outs on ISS.

1. Experiment Verification Test (EVT), Run 2

The second EVT was carried out in the Advanced Plant Habitat (APH) Ground Unit (S/N001) from 1/31/2018 to 3/14/2018.

Science Carrier Configuration and Plant Harvest:

• Each quadrant of the Science Carrier was filled with media consisting of approximately 1.6-1.7 L of washed and autoclaved 1-2 mm arcillite which contains 7.5 g/L of Type 180, 18-6-8 nutricote slow release fertilizer. Five to seven seeds each (in individual groupings) of A. thaliana WT, and transgenic adt5, adt4/5, adt3/4/5/6, WT/CCM, and adt3/4/5/6/CCM lines were adhered (using guar gum) to the gauze material in each of the 48 locations in the Science Carrier. The location of each line in each quadrant was randomly determined using a stratified randomized design.

• The Science Carrier was next installed into the APH Ground Unit (S/N001) and water was supplied to each of the four quadrants on January 31, 2018. Pictures were taken both from the side and the top of the APH and thereafter each day during the growth period.

• Fourteen days later (i.e., February 14, 2018), seedlings were thinned out so that only one remained in each spot). Prior to thinning, Pulse-Amplitude Modulated (PAM) measurements were carried on all seedlings using a FluorPen.

• After four-weeks growth (February 28, 2018), PAM measurements were carried on plants located in the front two rows of Quadrants 1 and 4 with the FluorPen and half of the plants in the Science Carrier (24) were harvested.

• The remaining plants were harvested on March 14, 2018.

• As before, for both harvests, plants were collected from the left to right and from the front to the back of the Science Carrier, separated into stem and rosette leaf samples, transferred into labelled foil packets, weighed, and then frozen at –150°C using a conditioned cold block in a mini cold bag. Following harvest of the last plant, specimens were transferred into a –80°C freezer where they were kept frozen until shipped to Washington State University (WSU) on dry ice, where they were again stored at –80°C.

Lignin Analyses:

• In order to determine if the Arabidopsis plants grown in the APH for the second EVT had attained the same level of maturity and lignification as those previously grown in the greenhouse at WSU, lignin compositions were estimated in a subset of plants. Thus, stems from WT, and mutants, adt5, adt4/5, and adt3/4/5/6, were freeze-dried, ground to a powder (in a mortar by means of a pestle), with cell wall residues next obtained as routinely performed in our laboratory. The cell wall residues of these selected plants were next subjected to thioacidolysis to estimate lignin amounts. Lignin levels were within the ranged observed in plants grown in the WSU greenhouse.

2. Experiment Requirements Document (ERD)

In collaboration with the Payload Development Team and upon completion of two Experiment Verification Tests (EVTs), the Experiment Requirements Document (ERD) was updated. On April 26, 2018, the ERD was presented to the SLPS (Space Life & Physical Sciences) Program Executive for Space Biology at NASA Headquarters who gave his approval to proceed with the flight experiment.

3. Flight Preparation

Two Science Carriers (SN003 and SN004) were prepared (April 26, 2018) by Kennedy Space Center (KSC) personnel as described above for EVT Run 2 and seeded on May 9 and 10, 2018. Again, the location of each line in each quadrant was randomly determined using a stratified randomized design for both Science Carriers.

These were then packed and shipped custom critical to Wallops, VA on May 11, 2018 to be loaded on resupply spacecraft Cygnus.Launch of Orbital ATK Antares rocket for OA-9 mission was on May 21, 2018 at 4:39 a.m. EDT. Cygnus connected with ISS on May 24, 2018 at 1:13 a.m. EDT. For the corresponding ground controls, two other Science Carriers (SN005 and SN006) were prepared in a similar way a week later.

4. First Grow-out on ISS

This was initiated by astronaut Ricky Arnold on June 8, 2018 (Day 0). While many seeds germinated, some sites showed no apparent germination. On June 22, 2018 (Day 14) FluorPen photosynthesis measurements were carried out, followed by thinning of the Arabidopsis seedlings to one per spot. Following thinning, several plants unexpectedly died. Overall, ISS APH Arabidopsis plants grew slightly slower than the ground control plants.

The consequence of spaceflight environment effects (reduced plant number, and somewhat smaller size, resulted in only one harvest at 45 days being possible, i.e., in order to ensure sufficient plant material for our multi-omics analysis. FluorPen photosynthesis measurements were, however, carried out after 31 days (4 weeks and 3 days) of growth (July 9, 2018). Plants were harvested on ISS on July 23, 2018, and immediately frozen and stored in the GLACIER freezer at –160 °C (as already done for the thinnings). A provisional explanation to the spaceflight environment/microgravity effect observed for the first grow-out was that the water introduced into the Science Carrier did not efficiently reach all 48 sites equally. This could have been caused by small bubbles in the water lines. Alternatively, it may have been partly due to intermittent partial lighting failures in the ISS APH.

This first set of samples was brought back to Earth (Splashdown in the Pacific Ocean, 01-13-2019), with transport and delivery to IBC/WSU on 01-15-2019). Our multi-omics analyses are now beginning. The ground control at KSC was initiated a week later (June 15, 2018; Day 0), with FluorPen photosynthesis measurements/thinning on June 29, 2018 (Day 14), FluorPen photosynthesis measurements on July 13, 2018 (Day 28) and final harvest on July 27, 2018 (Day 42). Plants were transported/delivered to IBC/WSU on 01-25-2019.

5. Second Grow-out on ISS

The second grow-out was initiated by astronaut Serena Auñón-Chancellor on September 18, 2018 (Day 0). The watering step was modified, with much improved germination occurring at 47 of 48 sites by 14 days.

Following FluorPen photosynthesis measurements, the subsequent thinning operation on ISS (October 2, 2018) apparently adversely affected growth/development of some Arabidopsis plants with several dying. This did not occur with KSC APH ground controls. FluorPen measurements were next carried out on October 19, 2018 (Day 31), with slightly slower growth again being observed in the ISS APH facility. To mitigate this, only a harvest at ~6 weeks (November 2, 2018; Day 45) was carried out. Thinnings and 6 week old harvested plants were immediately frozen and stored in the GLACIER freezer at –160 °C. These will provisionally be returned in late spring 2019. The ground control was initiated at KSC a week later (September 25, 2018; Day 0), with FluorPen photosynthesis measurements/thinning occurring on October 9, 2018 (Day 14), FluorPen photosynthesis measurements on October 26, 2018 (Day 31), and final harvest was on November 9, 2018 (Day 45).

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis; C allocation; water use efficiency (WUE), vascular plant growth/development; vasculature performance; auxin transport, and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi 'omics' effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome 'discrepancies' result from over-simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

2). We have partnered with Ms. Kathy Lucchesi (K-7/8 teacher), McCaffrey Middle School in Galt, California, and their largely Hispanic students. Supplemental funding was provided to the school by NASA and the California Space Grant Consortium so that these middle school students can safely follow and repeat many of the plant growth and development protocols developed for the International Space Station (ISS) experiments. One purpose here is that the students grow plants under similar conditions and obtain information and insights on how the research impacts or benefits life on Earth and beyond (in the future).

Dr. Lewis (Washington State University) and Dr. Sato (NASA Ames) visited the school on September 28, 2017, to see first hand the work underway. Both visitors also gave talks on the project, as well as the broader ramifications of NASA research.

The Galt Herald wrote an article on this visit ( http://www.galtheraldonline.com/news/mccaffrey-students-reach-for-the-stars-in-a-joint-experiment/article_b7047f9e-a93e-11e7-b016-d79e90b47bc5.html?utm_medium=social&utm_source=email&utm_campaign=user-share ), and students progress was further publicized through the NASA Space Biology Facebook ( https://www.facebook.com/spacebiology/ ).

Progress with the students is also followed through regular Skype and/or FaceTime team meetings, with Drs. Lewis, Davin, and Costa (Washington State University-WSU). Dr. Hanson (University of New Mexico-UNM) also provided tutorials on photosynthesis and the use of a FluorPen for their studies.

Written materials on, and seeds for, the experiments at hand are also routinely provided by WSU. The additional aim here is in helping teach and inspire these young students about the joys and fun of the scientific method in experimental plant biology. Periodically, the middle school students present results to Dr. Lewis over where such work is routinely evaluated.

3). A Pullman High School student (PHS Senior Charles Pezeshki) has also been involved in the WSU project. He carried out/refined Arabidopsis growth conditions using the Science Carrier that will be fitted into the Advanced Plant Habitat, APH. The Washington State Space Grant Consortium kindly provided supplementary support for both high school and undergraduate participation.

Mr. Pezeshki successfully completed his Senior Project, which led to several successful outcomes. He gave two posters (one at WSU for the University Showcase, and a second one in October 2017 in Seattle at the Annual Meeting of the American Society for Gravitational and Space Research (ASGSR) in a session for High School students nationwide). He was also supported by a travel grant from ASGSR to present his work.

Some of Mr. Pezeshki’s activities included:

i). Sieving, washing, and autoclaving of Turface arcillite particles used as the growing substrate for plants on board ISS, as well as preparation of foam and gauze templates to support the seeds and plants in the final assembly of the Science Carrier unit.

ii). Recording and editing videos to present to NASA Kennedy Space Center (KSC) personnel, so that they could review our consortium's plant harvesting and processing methods used for RNA, protein, and metabolite extraction. These videos were instrumental for assessing constraints involved in foil pack dimensions required for plant samples for adequate preservation during storage and for rapid processing upon return to Earth. This was helpful for developing procedures necessary for optimizing astronaut crew time spent on harvesting and critical storage of samples in the limited freezer container space aboard ISS.

iii). Assistance with transgenic plant sample preparation for PCR gene screening analysis. This involved DNA extraction from freshly harvested plant leaves, and use of these samples as DNA templates for the PCR mixtures. The samples were amplified using a thermocycler instrument and then run on an agarose gel for qualitative analysis assessment.

iv). Developing protocols for harvesting and processing different parts of Arabidopsis plants, which included separating leaves, stems, siliques, flowers, etc., and then later on grinding tissues and preparing extracts suitable for our multi-omics studies. He continuously assisted in preparing plant tissue samples for metabolomic analysis. He also prepared a video presentation on this that was also very helpful for the NASA scientists/astronauts by recording this entire process beginning from harvest to extraction.

v). Studying effects of humidity on metabolite levels at different harvest times. Mr. Pezeshki individually processed leaf and stem tissues of four and six weeks old Arabidopsis wild type plants grown under low and high humidity conditions. He studied the growth and development (stem length and weight) of Arabidopsis wild type plants harvested at different time intervals. He also successfully completed this experiment without assistance. After he had expertise in preparing tissue samples suitable for metabolomics, he was assigned to help study the metabolomics of NASA-SVT (Science verification test) samples.

• Establishing and compiling the Experiment Requirements Document (ERD) for consideration and approval by the SLPS (Space Life and Physical Sciences) Program Executive for Space Biology at NASA Headquarters.

• Carrying out the required Science Verification Test (SVT) and Experiment Verification Test (EVT). This was in order to test conditions that will be employed on International Space Station (ISS).

• All tasks were done in conjunction with the Payload Development Team (PDT) at Kennedy Space Center (KSC), and completed on April 24, 2017 and September 04, 2017, respectively, and with our consortium team partners, David Hanson (University of New Mexico), Mary Lipton (Pacific Northwest National Laboratory-PNNL), and Richard Sayre and Shawn Starkenburg (Los Alamos National Laboratory-LANL).

1. Experiment Requirements Document (ERD)

In collaboration with the Payload Development Team, an Experiment Requirements Document (ERD) was prepared. On March 14, 2017, the ERD was presented to the SLPS Program Executive for Space Biology at NASA Headquarters who gave approval for the SVT to be started. After completion of the SVT, a revised version of the ERD was again presented to the SLPS Program Executive for Space Biology on 7/13/2017. Upon approval, the EVT was then initiated.

2. Science Verification Test (SVT)

The SVT was carried out in the prototype Advanced Plant Habitat (APH) Engineering Development Unit at KSC. APH is a plant habitat, capable of hosting multi-generational studies, in which environmental variables (e.g., temperature, relative humidity, carbon dioxide level, light intensity, root zone moisture content, and spectral quality) can be monitored, controlled, and data recorded. For grow out of wild type (WT) and transgenic Arabidopsis thaliana lines (48 plants in toto), seeds were placed in a Science Carrier. The Science Carrier included sensors, manifolds, and watering tubes and is divided into four quadrants. Each quadrant was filled with arcillite containing a slow release fertilizer.

Science Carrier Configuration and Plant Harvest:

• Five seeds each of A. thaliana WT, and transgenic adt5, adt4/5, adt3/4/5/6, WT/CCM, and adt3/4/5/6/CCM lines were adhered (using guar gum) to the gauze material in each of the 48 locations in the Science Carrier. Location of each line in each quadrant was randomly determined using a stratified randomized design. This random design was chosen so that there will be two plants of each line within each quadrant.

• The Science Carrier was next installed into the prototype APH Engineering Development Unit (EDU) and water was supplied to each of the four quadrants on March 15, 2017. Pictures were taken both from the side and the top of the APH and thereafter each day during the growth period.

• Fourteen days later (i.e., March 29, 2017), seedlings were thinned out so that only one remained in each spot. Prior to thinning Pulse-Amplitude Modulated (PAM) measurements were carried on all seedlings using a FluorPen.

• After four-weeks growth (April 12, 2017), the Arabidopsis plants looked healthy; some of them had started to bolt. PAM measurements were carried out on plants located in the front two rows of quadrants 1 and 4 with the FluorPen, and half of the plants in the Science Carrier were harvested.

• Because of the amount of plant debris accumulating after 5 weeks growth, it was decided to harvest the remaining plants in 2 groups: Quadrants 1 and 4 on April 21 (at 37 days) and quadrants 2 and 3 on April 24, 2017 (at 40 days).

• For both harvests, plants were collected from the left to right and from the front to the back of the Science Carrier, separated into stem and rosette leaf samples, transferred into labelled foil packets, weighed, and then frozen at –150°C using a conditioned cold block in a mini cold bag. Following harvest of the last plant, specimens were transferred into a –80°C freezer where they were kept frozen until shipped to Washington State University (WSU) on dry ice, where they were again stored at –80°C.

Lignin Analyses:

• In order to determine if the Arabidopsis plants grown in the APH for 37 and 40 days had attained the same level of maturity and lignification as those previously grown in our greenhouse at WSU, lignin compositions were estimated in a subset of plants. Thus, stems from WT and the mutant adt3/4/5/6 were freeze-dried, ground to a powder (in a mortar by means of a pestle), with cell wall residues next obtained as routinely performed in the Lewis laboratory. The cell wall residues of these selected plants were next subjected to thioacidolysis to estimate lignin contents. Lignin levels were significantly lower in stems from WT and adt3/4/5/6 plants grown in the APH for the SVT as compared to those grown in the greenhouse for a similar time period. This was due to the plants not having grown and developed to the same extent.

3. Experiment Verification Test (EVT), Run 1

The first EVT was carried out in the Advanced Plant Habitat (APH) Ground Unit (S/N001).

Science Carrier Configuration and Plant Harvest:

• Five seeds each of A. thaliana WT, and transgenic adt5, adt4/5, adt3/4/5/6, WT/CCM, and adt3/4/5/6/CCM lines were adhered (using guar gum) to the gauze material in each of the 48 locations in the Science Carrier. As for the SVT, the location of each line in each quadrant was randomly determined using a stratified randomized design.

• The Science Carrier was next installed into the prototype APH Engineering Development Unit (EDU) and water was supplied to each of the four quadrants on July 27, 2017. Pictures were taken both from the side and the top of the APH and thereafter each day during the growth period.

• Eleven days later (i.e., August 6, 2017), seedlings were thinned out so that only one remained in each spot. Prior to thinning Pulse-Amplitude Modulated (PAM) measurements were carried on all seedlings using a FluorPen.

• After four-weeks growth (August 23, 2017), the Arabidopsis plants had grown slower than during the SVT, none had started to bolt, and a few did not grow in Quadrant 1. Again PAM measurements were carried out on plants located in the front two rows of Quadrants 1 and 4 with the FluorPen, and half of the plants in the Science Carrier (24) were harvested.

• The remaining plants were harvested on September 04, 2017.

• As before, for both harvests, plants were collected from the left to right and from the front to the back of the Science Carrier, separated into stem and rosette leaf samples, transferred into labelled foil packets, weighed, and then frozen at –150°C using a conditioned cold block in a mini cold bag. Following harvest of the last plant, specimens were transferred into a –80°C freezer where they were kept frozen until shipped to Washington State University (WSU) on dry ice, where they were again stored at –80°C.

Lignin Analyses:

• In order to determine if the Arabidopsis plants grown in the APH for the EVT had attained the same level of maturity and lignification as those previously grown in the greenhouse at WSU, lignin compositions were estimated in a subset of plants. Thus, stems from WT, and mutants, adt5, adt4/5, and adt3/4/5/6 were freeze-dried, ground to a powder (in a mortar by means of a pestle), with cell wall residues next obtained as routinely performed in our laboratory. The cell wall residues of these selected plants were next subjected to thioacidolysis to estimate lignin amounts. Lignin levels were significantly lower in stems from WT and adt3/4/5/6 plants grown in the APH for the SVT as compared to those grown in the greenhouse. Again this was due to the plants not having grown and developed to the same extent as at WSU.

4. Experiment Verification Test (EVT), Run 2

A second EVT was initiated on January 31, 2018. Its purpose is to modify plant growth parameters in the APH in order to attain Arabidopsis maturity/lignin levels comparable to those obtained in the WSU greenhouse for all WT and mutant lines. This work is ongoing.

5. APH Flight Unit Readiness Status on ISS

• The APH Flight Unit was launched in two shipments to the ISS on OA-7 (April 18, 2017) and SpaceX-11 (June 3, 2017). The on-orbit installation was completed on October 27, 2017. The first power-up was successfully completed on November 27, 2017 followed by a 5-day functional checkout which was successfully completed on December 01, 2017.

• In order to further test the ISS APH unit, a Science Carrier with WT Arabidopsis seeds in Quadrants 2 and 3 and dwarf wheat in Quadrants 1 and 4 was shipped to ISS.

• A Plant Growth Test was initiated with Quadrants 2 and 3 (Arabidopsis seeds) and Quadrants 1 and 4 (wheat seeds) successfully flood filled on January 22, 2018, and February 8, 2018, respectively.

• All specimens appeared to be growing well to date (as of February 28, 2018).

6. Sample Preparation for Metabolomics, Transcriptomics and Proteomics Analyses.

The overall scope of this proposal is to carry out transcriptomics, proteomics, and metabolomics analyses on leaves/stems of all plants harvested after 4 and 6 weeks of growth in the APH. Because the amounts of tissues recovered might be limited (~200 mg), we tested:

• An extraction method developed by PNNL allowing metabolites and proteins to be extracted from the same tissue (~150 mg) with these used for subsequent metabolomics and proteomics analyses. Twenty-four proteins samples were sent to PNNL for QC analyses: All passed. The corresponding metabolites samples were analyzed at WSU and also passed the QC test.

• An extraction method using ~50 mg tissue (fresh weight) to isolate mRNA for transcriptomics analyses. Forty mRNA samples were sent to LANL for QC analyses. All passed the QC analyses.

7. Seed Viability

In addition to the additional genetic transformations and establishment of requisite growth conditions etc. for the Arabidopsis, seed viability tests were carried out at WSU. These were studied because of the long lag time between shipping the two Science Carriers to ISS and the much later initiation of the experiments by flood filling the four quadrants.

Seed viability testing at WSU included:

• A Science Carrier prepared as before, with WT Arabidopsis seeds in the four quadrants.

• The first quadrant was flood filled, with all seeds germinating. After 2 weeks, the seedlings were thinned. All plants grew well the throughout the 6 week time period.

• The second, third, and fourth quadrants were each flood filled at a 6 week interval. As for Quadrant 1, all plants grew well.

• There were no problems noted with seed viability testing in the Science Carrier for at least 24 weeks.

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis, C allocation; water use efficiency (WUE), vascular plant growth/development; vasculature performance; auxin transport, and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi 'omics' effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome 'discrepancies' result from over simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

2). We are partnering with Ms. Kathy Lucchesi (K-7/8 teacher), at McCaffrey Middle School in Galt, California, and their largely Hispanic students on a program where elementary students can follow and repeat certain very safe plant growth experiments developed for the International Space Station (ISS) experiments, i.e., where we will assist on a “hands on” experience for the students, and their participation with the progress of our ISS multi-omics studies. This allows for the students to grow plants under similar conditions and to obtain information and insights on how the research impacts or benefits life on Earth. Our participation here includes offering instructions through Skype and written materials on the experiments at hand, and helping teach and inspire the young students about the joys and fun of the scientific method in experimental plant biology. Periodically, we will have these middle school students present results over Skype to our team meetings where such work is routinely evaluated.

3). Beginning September 2016, a high school student (Junior) has been involved in the project. He carried out/refined Arabidopsis growth conditions using the Science Carrier that will be fitted into the Advanced Plant Habitat, APH. The Washington State Space Grant Consortium is providing supplementary support for high school and undergraduate participation.

Approaches to the experimental design and implementation have thus focused on confirming the ability to successfully germinate and grow Arabidopsis for up to a 6-week harvest point in a reliable manner as previously done in other growth systems. This was required as a precedent to growing and harvesting the plants for experimental analysis, in order to prevent potentially confounding effects created by non-uniform environmental factors in the APH on ISS.

In conjunction with the KSC team, conditions were established to grow Arabidopsis in the newly developed APH, and which are considered potentially applicable for facile growth on ISS. This was developed based on numerous growth-out trials at Washington State University (WSU) over the last year to established best growth conditions, i.e., as regards water delivery, light intensity, humidity, etc.

The final growth out at KSC, which built up from the previous growth outs, was carried out into the APH Engineering Development Unit (EDU), and was initiated on January 10 and completed on February 21, 2017.

Photographs were taken each day of the Arabidopsis growth-out. Metabolomics analyses were carried out on these various growth-out samples to ensure that results obtained matched our earlier findings for greenhouse/growth chamber grown plants. Studies also examined different times of metabolite extraction of plant lines that had been stored at room temperature up to 90 minutes; metabolite profile traces examined were unchanged during the different time frames.

An Experiment Requirements Document (ERD) has been generated and will be submitted to NASA headquarters on March 8, 2017.

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis, C allocation; water use efficiency (WUE), vascular plant growth/development; vasculature performance; auxin transport, and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi 'omics' effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome 'discrepancies' result from over simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with 'low' resolution global subcellular system measurements (transcriptomics, etc.) through 'nth' dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.

2). We are partnering with Kathy Lucchesi (K-8 teacher), at Lake Canyon Middle School (collectively Lake Canyon) in Sacramento, California, as well as other teachers there, and their students on a program where elementary students can follow and repeat certain very safe plant growth experiments developed for the International Space Station (ISS) experiments, i.e., where we will assist on a 'hands on' experience at Lake Canyon for the students, and their participation with the progress of our ISS multi-omics studies. This allows for the students to grow plants under similar conditions and to obtain information and insights on how the research impacts or benefits life on Earth. Our participation here includes offering instructions through Skype and written materials on the experiments at hand, and helping teach the young students about the joys and fun of the scientific method in experimental plant biology. Periodically, we will have Lake Canyon students present results over Skype to our team meetings where such work is routinely evaluated.

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

2. Massa, G., Newsham, G., Hummerick, M.E., Caro, J.L., Stutte, G.W., Morrow, R.C., and Wheeler, R.M. (2013) Preliminary species and media selection for the veggie space hardware. Gravitational and Space Research 1: 95-106. http://gravitationalandspacebiology.org/index.php/journal/article/viewFile/616/636

We are very well positioned for incisive spaceflight and definition stage (1g) studies to investigate gene/metabolic network relationships and adaptations resulting from varying lignin and carbon assimilation levels, e.g., on photosynthesis, C allocation; water use efficiency (WUE), vascular plant growth/development; vasculature performance; auxin transport, and gravitational adaptations. Our overarching hypothesis is that a comprehensive interrogation (an integrative omics study) of our Arabidopsis lines with varying lignin levels and/or modulated carbon concentrating mechanisms (CCMs) or combination thereof will identify gene/metabolic networks, mechanisms and/or pathways that are differentially modulated at 1g and on exposure to microgravity, i.e., various omics (phenomics, transcriptomics, genomics, proteomics, metabolomics, and ICB) will allow us to study these in a truly unprecedented way.

Overall objectives:

1. Establish multi ‘omics’ effects of modulating lignin and CCM levels i) at 1g and ii) in spaceflight.

2. Compare/contrast data, using an ICB approach, to better define and understand gravity sensing and responses, and if threshold/induction parameters are modified or changed, when lignin and CCM levels are varied.

More specifically, we address distinct hypotheses for our various teams, and integrate, dissect, and incisively analyze them holistically in a manner hitherto not possible. These 5 hypotheses include that: modulating lignin and CCM levels differentially affect carbon assimilation/re-allocation, photosynthesis, and WUE (Team 1); modulating lignin and CCM levels differentially affect secondary and primary metabolite levels (metabolomics) (Team 2); system-wide modification in the transcriptome occurs through a common transcriptional regulatory mechanism, and transcriptome/proteome “discrepancies” result from over simplification of transcript analyses (Team 3); differential alterations in lignin and CCM levels can often be attributed to overall distinct changes in protein expression and phosphorylation patterns in a defined set of proteins (Team 4); an integrated omics analysis will provide urgently needed new insights into global effects on plant biological processes at both 1g and in microgravity (Teams 1-4). Each hypothesis draws upon the most advanced technologies available for study. We consider that our ICB approach will transform omics analysis through our advanced instrumentation and analytical tools. We will utilize (or design) computational tools/mathematical algorithms for integration and correlation of high resolution phenotype measurements (phenomics) with “low” resolution global subcellular system measurements (transcriptomics, etc.) through ‘nth’ dimensional analysis.

Our study aligns with Research Emphasis 1 and 3, and decadal survey elements in Cell, Microbial, and Molecular Biology (CMM-3, CMM-5), Organismal and Comparative Biology (OCB 2-5), Developmental Biology (DEV-4), and Plant and Microbial Biology, chapter 4 (P2). Our data generation will also be seamlessly integrated with various web-based platforms to handle, disseminate, and inter-actively utilize through iPlant and OpenMSI, and thus are made available to NASA as well as being a community resource.