NOTE: End date changed to 9/11/2020 per NSSC information (Ed., 9/18/19)

NOTE: End date changed to 9/11/2019 per NSSC information (Ed., 9/14/18)

NOTE: End date changed to 9/11/2018 per NSSC information (Ed., 12/13/17)

Insights from RNAseq analyses of APEX05 samples: Comparison of gene expression patterns in flight vs ground samples support the main hypotheses driving the experimental design of APEX05 that spaceflight induces both hypoxic and oxidative stress in plants. Thus, the wild type control plants show patterns of gene expression consistent with both hypoxia and oxidative stress. The hypoxia resistant cax2 mutants show less up-regulation of hypoxia-related transcripts, consistent with the idea that in these lines hypoxia responses are constitutively induced, allowing the plants to survive better once hypoxic conditions are established. The rbohD mutant also shows an altered pattern of oxidative stress-induced transcripts and some unique patterns of gene expression. For example, there was a significant increase in the differential expression of a cluster of loci involved in, e.g., defense signaling and ion transport.

Insights from GFP imaging: The imaging of the oxidative stress-responsive promoter:GFP construct from both flight and ground samples suggests rbohD plants show altered reactive oxygen species production in the roots on both the ground and during spaceflight. The imaging of this reporter system on-orbit in the LMM (light microscopy module) further suggests that there is no obvious change over the 24h of the imaging window in the development of signal from either the ROS- and stress- responsive promoter-reporter lines. These observations imply the oxidative stress related to flight has already built up over the time period of growth prior to imaging. These results further suggest that the RBOHD enzyme may be a critical element of spaceflight-induced oxidative stress responses. To further define whether this is indeed true, test are underway using hypoxic chambers and clinostat responses in these GFP bioreporter plants to see how closely these conditions mimic spaceflight response in wild type and how well the rbohD mutants resist these stresses.

Ground-based Analyses: In parallel to the APEX05 flight experiment, ground-based analyses have been pursued to complement the flight data. The critical experiments have been focused on delivering hypoxic stress to plants undergoing clinorotation to try and mimic the spaceflight environment as closely as possible. Patterns of growth in 2-d and 3-d clinorotated plants have therefore been followed with the 3-d clinostat most closely mimicking the growth patterns seen in spaceflight. High-quality, 3-d clinostats compatible with use in custom hypoxic chambers have been developed and to allow experiments where these stresses are combined to now be performed.

Studies of ground-based stress signaling systems in plants has also been pursued to place the spaceflight data in context. A previously undescribed long-range signaling system that plants use to communicate throughout the plant body has been uncovered by this research. The system is based around rapid, long-range, propagating changes in calcium levels within cells that carry stress information from one part of the plant to other organs that themselves are not experiencing the stress. The system is driven by the action of the Ca2+ channels of the glutamate-like receptor family. Characterization of this effect in response to a wide range of stresses from wounding to cold stress, hypoxia, and pathogen attack is underway.

Presentations and Outreach/Education: During 2018-2019, the APEX-05 project was presented at the 2019 MidWest Plant Cell Dynamics Meeting, the annual meeting of the American Society for Gravitational and Space Research, the ISS (International Space Station) Research and Development Conference and at venues such as the Academia Sinica in Taiwan. APEX-05 has also been presented at outreach-oriented events ranging from the University of Wisconsin sponsored outreach days (e.g., University of Wisconsin’s Science Expeditions) to presentations for high school students and undergraduates and middle school and K-12 teachers (such as at the Biotechnology Institute's summer teacher training program).

The laboratory maintains a “Collaboratory” where biology and engineering undergraduates and high school students come together to develop high throughput phenotyping equipment and other hardware relevant to our spaceflight-related work. Over 2018-2019 approximately 15 students have been mentored on various plant molecular and engineering projects related to our general space biology program and APEX05, with a large number working on bioinformatics-based projects driven by the APEX05 RNAseq datasets. This work also helps maintain a close collaboration with Dr. Andrea Henle at Carthage College, a 4 year liberal arts college in Wisconsin, where APEX05 analyses are an integral component of her space biology class.

This last year has also been notable with providing support with interviews and lab visits to about 20 groups of middle/high school students who are part of the First Lego League Championship. This year this league focused on challenges related to sustaining astronauts during long-term manned spaceflight. Many of the groups chose to design solutions for plant growth in space, hence their interest in projects such as BRIC-based studies and especially Veggie and experiments such as APEX05.

The lab has also been lucky to be able to share space/stress science with the local, national and international media through, e.g., interviews ranging from the local Badger Herald and The Sun Prairie Star newspapers through to the New York Times and National Geographic. We have also been fortunate to do a range of radio interviews with local radio stations such as Madison’s WORT and Wisconsin Public radio through to national distributors such as such as NPR (e.g., Science Friday and Big Picture Science).

NOTE: End date changed to 9/11/2019 per NSSC information (Ed., 9/14/18)

NOTE: End date changed to 9/11/2018 per NSSC information (Ed., 12/13/17)

Insertion of 26 plates into the Veggie and subsequent growth were nominal. Astronaut Scott Tingle took daily photographs of the 4 genotypes (wild-type plants and the cax2-2, cax2-3, and rbohD mutants) from plates mounted in the corners of the Veggie and at 4 and 8 days for the entire 26 plate set of the experiment. At day 8, the plates were photographed, opened, and the seedlings harvested into RNAlater containing Kennedy Fixation Tubes (KFTs). All samples were successfully harvested and the KFTs stored in the MELFI at -80°C prior to return.

Samples were returned successfully with a splashdown on January 13th, 2018. After successful de-integration of both the flight and parallel ground control materials, samples were shipped on dry ice to the university of Wisconsin-Madison for post-flight analysis by the PI team. Post flight analysis consists of:

1. Analysis of the on-orbit plant growth images. This analysis has been completed.

2. Imaging the samples using the confocal microscope to follow the dynamics of the RBOHDpromoter::GFP and Ubiquitin10promoter::mCherry signals. All the samples have now been successfully imaged and the imaging data is under analysis.

3. Isolation of RNA from samples dissected into root and shoots and analysis of gene expression patterns in flight vs ground samples using RNAseq is currently underway

Ground-based research: In parallel to the APEX05 flight experiment, we have continued to pursue ground-based analyses to complement the flight data. We have developed a series of hypoxic chambers where we can regulate O2 levels around plants. We have analyzed the response of wild type, cax2-2, cax2-3, and rbohD mutants to lowered O2 levels at the levels of both growth and qPCR analysis of marker gene expression.

We have also been analyzing ~20 genes targeted from an analysis of spaceflight transcriptomics data as being both up- or down-regulated in spaceflight and related to hypoxia and/or ROS-related signaling in ground-based research. This work is now showing an unexpected relationship between spaceflight response genes and effects on gravity signaling on Earth, suggesting that some of the spaceflight transcriptome changes may reflect disruption of a gravity sensing and response network of elements not predicted from classic gravitropism analysis on Earth.

Presentations and Outreach/Education: During 2017-2018, we have presented the APEX-05 project at the 2018 MidWest Plant Cell Dynamics Meeting, the annual meeting of the American Society for Gravitational and Space Research, and the American Society of Plant Biologists and at universities, such as Carthage College. We have also used APEX-05 as a base for our outreach efforts, where we have presented it at events ranging from University of Wisconsin sponsored outreach days (e.g., University of Wisconsin’s Science Expeditions) to presentations for high school students and undergraduates (e.g., BioHouse and summer undergraduate research programs) and middle school and K-12 teachers (Biotechnology Institute summer training program) and to Madison’s Boys and Girls club.

We are also working closely with Madison West High School’s Rocketry Society. These students have designed, built, and flown Arabidopsis experiments on their rocket flights, including now multiple NASA-sponsored launches at Marshall Space Flight Center. We have then been able to train them in plants science analysis such as phenotyping and molecular analyses (qPCR) of their rocket flown samples. This is a continuing program where we are developing a pipeline of talented and engaged students who are going on to College in STEM (Science, Technology, Engineering, and Math) areas.

We maintain a “Collaboratory” where biology and engineering undergraduates and high school students come together to develop high throughput phenotyping equipment and other hardware relevant to our spaceflight-related work. Over 2017-2018 we have mentored approximately 10 independent study students working on various plant molecular and engineering projects related to space biology and APEX05. We also run a space biology related practical lab course called AstroBotanical Engineering. Here a pool of plant biology and engineering students collaborate to develop space-related hardware for ground-based testing of plant science space-related projects. We have ~15 students per semester. We are also working with Dr. Andrea Henle at Carthage College, a 4 year liberal arts college in Wisconsin to help use APEX05 and space plant biology as an integral component of her new space biology class.

We have also been fortunate to have interest in this research from several media organizations and so we have participated in a series of interviews with groups ranging from local newspapers such as the Badger Herald and local and national radio (Wisconsin Public Radio, WORT radio, and Greensense radio), to podcasts (e.g., EdgeEffects and Founders Fund) to outreach venues such as Madison’s Space Place, Soundwaves, and Science on Tap.

We have also extended our analysis of the utility of far-red irradiation as a means of delaying seed germination to screening a broad spectrum of target crops/cultivars that may potentially be used with the Veggie. Seeds are currently being assayed over 6 months for efficiency and duration of germination suppression.

Flight Readiness: Both the Science and Experiment Verification Tests have been successfully completed during 2015 and 2016, meeting the “excellent” level for all success criteria. The experiment has passed Flight Readiness Review and is scheduled for launch on SpaceX13.

Ground-based Analyses: In parallel to preparing for flight, we have developed a series of hypoxic chambers where we can regulate O2 levels around plants. We have been testing the response of Arabidopsis to lowered O2 levels to develop a database to compare the APEX-05 flight materials to. QPCR analysis of marker gene expression suggests that some of the transcriptional fingerprints of genes upregulated in spaceflight can be mimicked at 10% O2 or lower. For example, induction of HSP101 and HSP70 occurs as seen in spaceflight samples at 6% O2. However, hypoxic challenge on Earth does not mimic all aspects of spaceflight. For example, spaceflight repressed genes such as the peroxidase superfamily are not repressed by hypoxia.

Transcriptional fingerprints from spaceflight data also strongly suggest reactive oxygen species stress is part of the plant responses to spaceflight. Treatment of Arabidopsis with short-term/acute oxidative stress from addition of H2O2 does not mimic spaceflight responses as assessed by QPCR analysis of marker genes. However, we postulate that the spaceflight environment is more likely delivering long-term (many days), low levels of oxidative stress. Long-duration H2O2 application is complex as the H2O2 degrades rapidly when added to plant samples. We have addressed the long-term treatment challenge in two ways. We have added H2O2 to plants at 2 day intervals during their growth and also developed a system to apply continuous H2O2 application to plants growing on Petri dishes. This 3-d printed ‘ROS-flow’ system provides a laminar flow of H2O2 solution over the face of a Petri dish where the plants are growing and is allowing us to monitor response to plants experiencing constant but low level H2O2 application.

Mutant Analyses: We are analyzing ~20 genes targeted from an analysis of spaceflight transcriptomics data as being both up- or down-regulated in spaceflight and related to hypoxia and/or ROS-related signaling in ground-based research. These include a range of heat shock-related, Ca2+ signaling and ROS-related genes such as AtRBOHC, D and F. We have isolated homozygous knockout mutants in these genes and are phenotyping them for alterations in ROS, hypoxic, touch, and gravitropic response. These analyses are revealing previously unknown links between spaceflight responsive transcriptome and signaling pathways thought to be altered in the microgravity environment. For example, we have found that mutants in HSP family members that are upregulated in spaceflight but classically linked to molecular stress responses, have significant defects in gravitropic response in the root. Thus, this analysis targeting spaceflight transcriptomics is now also helping increase the molecular details of the gravitropic response machinery.

Presentations and Outreach/Education: During 2016-2017, we have presented the APEX-05 project at the 2017 MidWest Plant Cell Dynamics Meeting, the annual meeting of the American Society for Gravitational and Space Research, and the American Society of Plant Biologists. We have also used APEX-05 as a base for our outreach efforts, where we have presented it at events ranging from University of Wisconsin sponsored outreach days (e.g., UW’s Science Saturdays and Science Expeditions) to presentations for high school students and undergraduates (e.g., BioHouse and summer undergraduate research programs) and K-12 teachers (Biotechnology Institute summer training program). We have also used large attendance opportunities such as the Madison Garden Expo to perform space biology-related outreach to the general public. We have also had the opportunity to talk about APEX-05 and space biology internationally, e.g., through Skype interviews with high school students in the UK and will be presenting at Sir Isaac Newton College in the UK this summer. Dr. Barker, a scientist in the lab working on APEX-05 also presented the APEX-05 work at the Chinese Academy of Science/National Academy of Science Forum for New Leaders in Space Science

We are also working closely with Madison West High School’s Rocketry Society. These students have designed, built, and flown Arabidopsis experiments on their rocket flights, including NASA-sponsored launches at Marshall Space Center. We have then been able to train them in plants science analysis such as phenotyping and molecular analyses (QPCR) of their rocket flown samples. This is a continuing program where we are developing a pipeline of talented and engaged students who are going on to College in STEM areas.

We are also blending our biology-based plant space science with educational experiences for both local high school and college students (including those who may not have been initially attracted to biological sciences). Thus, we have mentored the UW-Madison introductory engineering class over the course of the 2016/2017 academic year to develop practical solutions to projects related to space science and the phenotyping needed to understand the results from APEX-05. For example, this Spring semester the students were tasked with the development of 3-d clinostats and acoustic levitators to grow plants in an environment lacking physical contact with a growth medium. Many of these students continue with these projects by working in the lab for credit as independent study students or enrolling in the AstroBotanical Engineering class (see below).

We also maintain a “Collaboratory” where biology and engineering undergraduates (and high school students) come together to develop high throughput phenotyping equipment and other hardware relevant to our spaceflight-related work. We mentor approximately 10 independent study students working on various plant molecular and engineering projects related to space biology. This interdisciplinary approach has also allowed us to establish a space biology related practical lab course called AstroBotanical Engineering. Here a pool of plant biology and engineering students collaborate to develop space-related hardware for ground-based testing of plant science space-related projects. We have 15-20 students.

We have also been fortunate to have interest in this research from several media organizations and so we have participated in a series of interviews with groups ranging from local newspapers such as the Badger Herald, to space-oriented podcasts (e.g., L9).

The experiment will use 4 different genotypes of Arabidopsis: wild type, the cax2-2 and cax2-3 mutants, and a mutant in the gene AtRBOHD. CAX2 is an ion transporter that previous analysis has shown likely plays a critical role in signaling of hypoxic stress. RBOHD is an enzyme that produces reactive oxygen species in response to a wide range of environmental cues. The mutant plant lines for these studies have been generated and the project has moved to testing and validation in preparation for a flight opportunity.

Both the Science and Experiment Verification Tests (SVT and EVT) have been successfully completed during 2015-2016 in order to troubleshoot and test the experimental design and procedures. The SVT was performed February-March 2016 and results met the “excellent” success criteria for all elements of the experiment, indicating the experimental approaches and procedures were likely sufficient to support a flight. Therefore, an EVT was conducted May-July 2016. The results from EVT again met the “excellent” level for all success criteria. The far-red irradiation effectively delayed germination in all samples and once triggered to germinate by the lights of the Veggie hardware, seedling growth was efficiently initiated in all samples. At the end of the experiment, samples were chemically fixed and frozen and subsequently RNA was isolated in sufficient quantities and with sufficient purity to perform the RNAseq gene expression analysis that is one major focus of this research.

The lines used for these experiments have also been engineered with green fluorescent protein (GFP)-based reporter for monitoring ROS response. Therefore, prior to isolation and analysis of RNA, GFP imaging will be performed on flight and ground samples. GFP fluorescence can be maintained despite fixation of the samples in RNAlater and freezing at -80 degrees and so all EVT samples were imaged to assess GFP signal after fixation and freezing. All samples showed preservation of fluorescence signal to the levels required for analysis of cellular distribution.

In parallel to preparing for flight, research has been progressing on defining the ROS-based signaling system in Arabidopsis and analyzing the effects of genes targeted as being related to hypoxia and/or ROS-related signaling from analysis of previous spaceflight transcriptomics data. These genes include Ca2+-signaling–related genes, molecular chaperones, and oxidative stress-related genes. The knockout mutants in these genes have been verified and are now being tested for alterations in responses linked to spaceflight, such as changes in gravitropic growth and sensitivity to hypoxia and ROS (H2O2) treatment. In collaboration with Dr. Richard Morris at the John Innes Centre, Norwich, UK, work has also been conducted on modeling how ROS and Ca2+ interact to propagate stress responses in plants. Using a combination of in vivo imaging of Ca2+ and ROS dynamics and analysis of responses in the AtRBOHD mutant, the role of this enzyme in ROS-assisted Ca2+-dependent stress signaling has been dissected. This quantitative analysis allowed Richard Morris to develop a math-based model to describe this phenomenon. The model predicts a role for AtRBOHD-dependent extracellular ROS production in cell-to-cell movement of stress information within the plant. Using measurements of responses to inhibitors of AtRBOHD, mutants, and quantitative imaging, the predictions of the model have been tested and the model validated.

APEX-05 research is also being used as the core of a suite of outreach efforts ranging from participation in local University sponsored outreach days (e.g., UW’s Science Saturdays and Science Expeditions) to presentations at Botany and Astronomy clubs and hands-on programs in adult education (e.g., the DIY Science program). Educational experiences for both local high school and college students (including those who may not have been initially attracted to biological sciences) are also being offered. Thus, UW-Madison’s introductory engineering class has been mentored over the course of the 2015/2016 academic year to develop practical solutions to projects related to space science and the phenotyping needed to understand the results from APEX-05. For example, the students were tasked with the development of 3-D clinostats and slow speed plant growth centrifuges. Partnering with Washington University in St Louis is now being explored to extend this program to involve design and architecture students and so broaden the multidisciplinary nature of this opportunity. This interdisciplinary approach has also allowed the establishment of the Astro-Botanical Engineering Society (ABES) based at the Wisconsin Institute of Discovery Center as part of the Wisconsin Outreach Collaborative (WOC). The group mentors approximately 20 regularly participating students, ranging from engineers, computer scientists, and mathematicians to botanists. These students also focus on generating solutions for the large scale phenotyping and measurement systems to support APEX-05-related analyses.

For this APEX-05 experiment, we plan to analyze mutants in a gene linked to hypoxic signaling (cax2) and in a major ROS producing enzyme (AtRBOHD). We plan to use a fixation/imaging protocol for green fluorescent protein (GFP) that will allow us follow the dynamics of this ROS response with high spatial resolution in the spaceflight materials. The major goal of the initial period of this work has therefore been to develop the genetic tools to support this last goal of being able to image GFP-expressing reporter lines.

Making Transgenic Reporter Plants:

In order to visualize ROS response throughout the plant in space-flown plants, we will use plants transformed with a GFP reporter system driven by a ROS-inducible promoter. The ROS-responsive promoter is that from the AtRBOHD gene. In order to make this a quantitative assay, we also plan to use a constitutive promoter (from Ubiquitin 10, UBQ10) driving a different GFP (mCherry, a red fluorescent version). The ratio of signal between GFP and mCherry will then provide a measure of ROS response independent of any variation between plants or individual organs in their general levels of protein production. Over the reporting period, we have cloned the requisite genes/promoters and are currently generating transgenic reporter lines in the model plant Arabidopsis expressing these constructs.

Supressing Germination:

This experiment requires Arabidopsis seeds to be planted at Kennedy Space Center but to germinate on orbit in the VEGGIE aboard the ISS. To delay germination of plants until they are in the VEGGIE hardware we have been testing the far-red light germination suppression system developed in the Blancaflor lab for APEX-03. This involves far-red light irradiation of seeds planted in Petri dishes using a 730 nm LED and then storage of the irradiated seed in darkness. Germination is subsequently induced by moving the Petri plates to white light, such as provided by the VEGGIE. Testing indicates this system can robustly delay germination for at least 2 weeks and our current analysis is testing the reliability of germination delays well beyond this period. At a practical level, testing is showing that manually wrapping the Petri dishes in foil and then placing these in a plasticized-foil bag provides a robust method of maintaining the plates in darkness after irradiation with a level of protection compatible with the handling needed for spaceflight. To increase throughput for the far-red irradiation step, we have also partnered with the Mechanical Engineering Department at UW-Madison and as part of their introductory engineering program (InterEGR 160), we have had 2 groups of undergraduate students design automated irradiation systems that will handle 6 Petri dishes at a time and automatically load them into the foil bags at the end of the treatment. They have delivered 2 prototypes for the equipment and these are currently under testing.

Mutant analyses:

We have also targeted ~20 genes from analysis of our transcriptomics data from previous spaceflights as highly likely to be related to low oxygen and/or ROS-related signaling in spaceflight. These include a range of heat shock-related proteins and ROS-responsive genes such as AtRBOHD. We are isolating homozygous knockout mutants in these genes and are currently assaying these mutants and wild-type controls for their response to different levels of O2 using growth and assaying the level of genes known to be changed in spaceflight as our measures of response.

Presentations and outreach:

During 2015, we have presented the spaceflight hypoxia project at the MidWest Plant Cell Dynamics Meeting in Madison, the International Space Station Meeting in Boston, the Plant Oxidative Group meeting in Verona, the Plant Stress meeting in Heidelberg, and the Plant Pathology student and postdoc seminar series at UW Madison. We plan to present at the meeting of the American Society of Plant Biologists in Minnesota in July and at the ASGSR meeting in November. As part of our space science outreach we have held events at the Westside Christian High School, Sigma Alpha Iota music society and the UW Health Center. We have also established a Facebook page: < https://www.facebook.com/pages/The-Gilroy-Lab/1393946517599092 > in preparation for beginning the active flight component of this grant.