The concomitant ground and flight support represented by this type of grant enables us to combine the processes of flight biology preparation, flight data analysis, and hypothesis development and testing for unprecedented insight into biological responses to these novel environments. The execution of this grant was (and continues to be) the ideal example of what NASA intended as the role of Fundamental Space Biology Ground-Based Research: NRA-01-OBPR-06 “…to expand our understanding of fundamental biological processes and the mechanisms by which these processes sense, respond, adapt to, and evolve in the space environment…and in particular… the role of the genome and cellular structures in sensing and responding to gravitational force.”

During spaceflight, plants experience growth and developmental conditions that are distinctly unlike the terrestrial environment that has directed the evolution of gene expression patterns that ensure survival. Plants must therefore interpret novel stimuli, integrate new solutions and express potentially unusual suites of genes in an attempt to cope with spaceflight environments. Some of these responses might be directly interpretable as appropriate responses that clearly inform science of the stresses induced by spaceflight. Other responses might be confused or inappropriate due to the novel signals or biological processes present in the milieu of the spaceflight environment. Defining both kinds of responses is critical to understanding biological response and adaptation to all kinds of extraterrestrial environments, from vehicles in orbit to lunar habitats.

Our objective is to answer fundamental questions regarding the metabolic and molecular response of plants to novel, spaceflight relevant, environments. In the current project we build on extensive insights gained from spaceflight and analog experiments to develop new biological tools and to guide hardware development to study the response to these environments with higher fidelity and wider scope. The biology will include new biosensors created to dissect the mechanisms by which molecular signals are conveyed and integrated into responses to spaceflight environments. Some biosensors will be linked to Green Fluorescent Protein (GFP) spectral variants to facilitate telemetric data collection, which will in turn enable robust robotic experiments with wide deployment options. The hardware to support this telemetric science, will be advanced in concept to include multiple spectral capacities in order to monitor multiple biosensor reporter genes simultaneously. The focus is on advancing telemetric imaging that will enable the potential future deployment of such biosensor-units in a wide range of potential spaceflight applications - ISS, crew vehicles, satellites and lunar or Mars packages - as the opportunities present. These experiments address the need to understand the biological response of eukaryotes to spaceflight, and other novel environments at the molecular and cellular level, with special focus on signaling pathways and gene expression.

Summary

Focus and Opportunities

The final year of the grant continued to help support people and efforts for additional characterizations of returned Flight materials from the PIs’ two Flight Experiments: APEX-TAGES and BRIC-16, as well as contributing to the support of ancillary analyses associated with the BRIC-17 returned material. Space Biology support enabled an additional level of analyses of these samples, and facilitated supplemental ground studies that continue to contribute to our understanding of the response of biology to spaceflight and novel environments. The data analyses led to the design and support of several analog studies initiated last year: 1) clinorotation and gravity vector disruption, including root skewing analyses in inclined planes, 2) hypergravity tests with a 2-3g centrifuge, and 3) a parabolic flight campaign opportunity and suborbital flight preparations in high performance aircraft (in support of two FO awards).

The concomitant ground and flight support represented by these grants enables us to combine the processes of flight biology preparation, flight data analysis, and hypothesis development and testing for unprecedented insight into biological responses to these novel environments. The execution of this grant was (and continues to be) the ideal example of what NASA intended as the role of Space Biology Ground-Based Research: NRA-01-OBPR-06 “…to expand our understanding of fundamental biological processes and the mechanisms by which these processes sense, respond, adapt to, and evolve in the space environment…and in particular… the role of the genome and cellular structures in sensing and responding to gravitational force.”

The final year of the grant had several foci:

Undifferentiated cells - cell sensing and signal transduction: The results of the BRIC-16 experiment (see FSB 2012 report and related BRIC-16 report) revealed that undifferentiated cells can detect the spaceflight environment and altered gravity in the absence of differentiated tissue or organized developmental structures (Paul et al. 2012: BMC Plant Biology. 12:232 http://www.biomedcentral.com/1471-2229/12/232 , Zupanska et al. 2013: American Journal of Botany. 100:235-248). One of the most striking results was the engagement of “heat shock” and protein chaperone genes The results from the tissue culture experiments leads us to further dissect the sensory and signal mechanism(s) that cell cultures use to detect and respond to spaceflight. To further address these questions, we started a series of ground experiments to test signal transduction pathways in unit gravity, and to also stage out biological tools for future flight opportunities. We initiated several new tissue culture lines, some of them containing GFP reporters, and others that are deficient in key chaperone genes. Experiments with cultures carrying known mutations that are well characterized in the gravity sensation and signaling of intact plants will provide insight into the mechanisms behind those processes. If cell cultures are using these typical sensing and signaling elements, the mutations should greatly attenuate the cell culture responses

Reporter gene line development: The results of the APEX-01 / TAGES and the BRIC 16 experiment revealed that a large number of genes were differentially expressed in response to the spaceflight environment (Paul et al. 2012: BMC Plant Biology. 12:232 http://www.biomedcentral.com/1471-2229/12/232 ; Paul et al 2013: BMC Plant Biology. 13:112 http://www.biomedcentral.com/1471-2229/13/112 ). These results initiated the development of a variety of new GFP-reporter gene lines to support spaceflight applications.

Biosensors in calcium-signaling mutant backgrounds: A number of reporters were developed in calcium signaling mutant backgrounds; two lines show differential expression of the reporter gene in plants deficient in aspects of calcium signaling.

Quality testing of flight hardware: This grant helped to support the bio-compatibility testing of two types of spaceflight hardware under consideration, the Aluminum PDFUs for future BRIC experiments, and a new ceramic foam sponge for the ABRS plant biology growth chamber.

Parabolic flight campaigns (Zero-G): This Space Biology grant contributed to the support of several parabolic flight campaigns that were awarded through the Flight Opportunities Office. Since FO does not provide operations support, it is essential that the PIs have some other source of NASA funding to support the personnel and analyses. Our NASA NNX09AL96G grant was the foundation of this support, and crucial for our success.

Performance aircraft for Sub-orbital application: The other un-supported Flight Opportunity Award received by the PIs is for a series of suborbital flights. This Space Biology grant also contributed to the support of preparations for the suborbital FO by conducting hardware and biology tests in high performance aircraft (T6 and F104).

Concluding remarks: We have accomplished an amazing amount of diverse, spaceflight-related science with this grant. Having this support has allowed us to develop a large range of biological and engineering tools to support four flight experiments, three parabolic flight campaigns, and enhance the technical readiness of our upcoming suborbital flight opportunity – all while conducting novel, fundamental ground-based research that expands the communities understanding of the molecular mechanisms associated with the spaceflight acclimation of plants. This research funding contributed to 16 publications over its lifetime, and to the support of three PhD students (Anne Visscher, Eric Schultz, Natasha Sng), three undergraduates (Lauren Frizzle, Justin Goodwyn, Zackary Guignardi), four postdoctoral associates (Agata Zupanska, Fiona Denison, Matthew Bamsey, Thomas Graham), and two biological scientists (Claire Amalfitano, Lawrence Rasmussen).

The concomitant ground and flight support represented by these grants enables us to combine the processes of flight biology preparation, flight data analysis, and hypothesis development and testing for unprecedented insight into biological responses to these novel environments. The execution of this grant was (and continues to be) the ideal example of what NASA intended as the role of Fundamental Space Biology Ground-Based Research:

NRA-01-OBPR-06 “…to expand our understanding of fundamental biological processes and the mechanisms by which these processes sense, respond, adapt to, and evolve in the space environment…and in particular… the role of the genome and cellular structures in sensing and responding to gravitational force.”

Some details include:

Extensive optimization of RNA analyses for all research in the laboratory, including utilizing BioAnalyzer technology for evaluations of RNA integrity.

Seed lines - In 2011, several of the lines characterized in 2010 were included in the Flight opportunity: pTCS::GFP, ARR-family mutant, AXR3 and EIR1. Those data are currently being analyzed.

Verification of calcium signaling mutations - the verification of mutants is to confirm that the Arabidopsis lines carry the T-DNA insertion, and that it is positioned to abolish gene function. The presence of the pBIN-pROK2 insert was verified using a set of primers derived from the sequence found on the Salk website ( http://signal.salk.edu/pBIN-pROK2.txt-new ). The location of the insert was verified with a primer derived from the genomic DNA of Arabidopsis upstream of the left insert flanking region (sequence provided by TAIR- arabidopsis.org) and LBa1, provided by the Salk site. DNA was isolated form mutant and wild type plants and used as templates for amplification with primers to regions within the coding region of the target gene and to sequences unique to the T-DNA insertion. Amplified sequence form the mutant plants (CBL1, CBL9, CIPK3, CIPK23) contain discrete amplified sequences corresponding to the T-DNA insert, whereas the wild-type (WT) does not. Amplification products confirm that the genes encoding the calcium signaling genes of interest in the mutant plants is disrupted by the T-DNA insertion.

Production of Biosensor plants in mutant backgrounds - Once verified, the calcium signaling mutants are used as recipients for pollen from plants homozygous for several reporter gene constructs. Cross pollination is carried out by selecting flowers whose sepals have not yet opened, at flower stage 10. The stamens and part of the petals and sepals are removed to prevent self-pollination. At flower stage 11, they are pollinated with stamen from one of the GFP reporter-gene donors, then covered with a small piece of plastic wrap from prevent the pistil from drying or being contaminated with any other pollen source. Currently we have completed six of the 12 targeted lines.

Phosphorylation mediated signal transduction - The involvement of phosphorylation events in signal transduction also appear to be strongly involved is a variety of stress responses pathways, and we are exploring phosphorylation-mediated events in addition to the calcium mediated events. The initial experiments with T-DNA insertion mutants examined the impact of the mutation on the expression of a number of genes associated with abiotic stress that involves a phosphorylation-mediated step in its pathway. The majority of these experiments were conducted by Dr. Fiona Denison, Dr. Agata Zupanska, and Mr. Justin Goodwyn (undergraduate). Thus far, seven different target genes and eight different phosphorylation mutants have been evaluated.

Parabolic Flight Data Analyses - In addition to the conducting of new experiments salient to the current grant, the grant also supported new data analyses of parabolic flight data collected under a previous award. Briefly, in a succession of experiments on NASA’s KC-135 and C-9 parabolic aircraft, Arabidopsis plants were exposed to a series of flight sets that provided controlled, replicated exposure to parabolic flight. Gene expression profiling of these plants reveals that parabolic flight, each presenting 40 durations of alternating microgravity and hypergravity, can induce wide-scale changes in gene expression patterns that stand the statistical tests of replication on three different flight days. The earliest response is characterized, after 20 parabolas, by a prominence of genes primarily associated with signal transduction. After 40 parabolas this prominence is largely replaced by a variety of genes associated with biotic and abiotic stimuli and stress. Among these responses, three metabolic processes stand out in particular: the induction of auxin metabolism and signaling, many of which are known to be involved in gravity sensing in plants; the differential expression of genes associated with calcium mediated signaling, and the repression of genes associated with disease resistance and cell wall biochemistry. Changes in auxin-related gene expression were also recorded by reporter genes tuned to auxin signal pathways. These data demonstrate that the parabolic flight environment is appropriate for molecular biology research involving the transition to microgravity, in that with replication, proper controls and analyses, gene expression changes can be observed in the timeframes of typical parabolic flight experiments. These analyses facilitated the publication of those data in this period, and the manuscript is currently under review in Astrobiology.

RNA Calibrations - -

High quality RNA is essential starting material for analyses of gene expression. A series of experiments were conducted to calibrate efficacy of several harvest and storage scenarios. An example of an experiment set is described in Table 1 and Figure 1 described in the Annual report. Table 1 and Figure 1 illustrate that the source of the material has a significant impact on the efficiency of RNA recovery. RNA was isolated from light-grown Arabidopsis seedlings (whole plants, dissected roots and dissected leaves) and compared to dark grown (etiolated) seedlings and cultured cells. The relative amount of RNA in etiolated seedlings was about 8-fold less than as was found for light-grown plants.

Seed lines - -

A number of Arabidopsis seed lines are available at a minimal cost to academic researchers from the Arabidopsis Information Resource (TAIR). A number of seed lines were obtained for their morphological phenotypes in response to alterations in gravity sensing, and these are under various stages of characterization. In an experiment testing reporter gene expression in response to changes in the gravity vector, plants were grown vertically on nutrient agar plates, then at 5 days old were turned 90 degrees clockwise. Plants were harvested to x-gluc stain (to visualize tissue-specific transgene expression) and fixed and photographed with a dissecting binocular microscope. In addition to the TAIR lines, there are several reporter gene lines under construction.

Telemetric Data collection - COTS Web cams for white-light imaging. - -

A design for a telemetry system using webcams and Active Webcam software was developed and is being tested for following plant growth and development remotely. The goal was to find a camera system with excellent resolution while keeping an economical mindset. The system that was designed allows for easy integration for multiple webcams being setup for a telemetry system. The system was tested and a time lapse video was created simulating the results of the experiment over a period greater than three weeks. The system is being increased on versatility and system integrations. LABVIEW will be used in the upcoming prototype along with the creation of a field point system.

NASA-developed Fluorescent Imaging Hardware - -

Over the past several years we have worked with KSC scientists and engineers to test the efficacy of several versions of a GFP imager, representing a development path toward units that could be deployed in shuttle and ISS systems, as well as satellites, landers and habitats. This past year (2009) has seen the culmination of those efforts with a finalized version of the GFP Imager; a unit of which was launched as part of an ongoing flight experiment on STS-129. This unit allows for images that can be captured and delivered telemetrically from a remote location. This hardware will also be used for the ground based, analog environments, which are the focus of this project.