NOTE: End date changed to 09/15/2019 per NSSC information (Ed., 3/12/19)

NOTE: End date changed to 3/11/2019 per NSSC information (Ed., 9/14/18)

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

NOTE: End date changed to 9/11/2017 per NSSC information (Ed., 6/14/16)

Expression profiling revealed a large number of Bd21 genes whose expression either increased or decreased in shoots and/or roots under microgravity. Most of the differentially expressed genes were organ-specific, and did not respond to either GS or MS under ground-control conditions. The lists of microgravity-response genes were enriched for genes predicted to function in plant responses to environmental and oxidative stress as well as reactive oxygen species. They were also enriched for genes involved in radiation response, implying that Brachypodium might serve as a good model to investigate the effects of cosmic radiation on plants. Few of the microgravity-response genes were also differentially expressed in response to GS or MS on the ground. Those that did respond to both GS and microgravity constitute excellent candidates for a function in gravity signal transduction. Overall, our studies identify several genes and pathways that could be further engineered to improve monocot plant adaptation to the microgravity environment, facilitating their use in bioregenerative life support systems for long-term space-exploration missions.

Ground based studies were also carried out to investigate the natural variation that exists between Brachypodium accessions for root-growth and behavioral responses to GS. These studies led to the discovery of several key loci that may contribute to the regulation of proprioception in plants.

Experimental Results and Conclusions

Development of a plant growth chamber to test Brachypodium distachyon seedlings growth under microgravity conditions

To test the ability of Brachypodium distachyon seedlings to germinate, grow, and adapt to the microgravity environment of the ISS, we developed a novel plant growth unit (named APEX Growth Chamber) in collaboration with Dr. Howard Levine and the NASA Kennedy Space Center Flight Support team. This system is based on an Oasis foam-based growth system previously used by Dr. Levine to grow plants in space. With this setup, Brachypodium seeds are inserted pre-flight into a dry block of foam that is surrounded by gauze and a nylon mesh, and is mounted on a medium injection device within a Magenta-box assembly. The seeds are kept in a dormant dry state, unable to germinate during spaceflight to the ISS. When the experiment is ready for activation on ISS, an astronaut can easily inject liquid growth medium into the foam support through the injection device, thereby imbibing the seeds and triggering their germination under red light.

This novel plant germination and growth system had to be developed because early preparatory experiments had demonstrated that the far-red light pretreatment typically used to prevent the germination of Arabidopsis thaliana seedlings on agar-based media during takeoff and travel to the ISS, has an opposite effect on agar-medium-embedded Brachypodium seeds, triggering their germination. An alternative light-based inhibition protocol we developed to inhibit Brachypodium seed germination during spaceflight could not be implemented because it required continuous seed exposure to blue-light and cold, thereby requiring excessive amounts of energy during a sensitive period of spaceflight. Our flight experiment and corresponding ground control involved a total of 15 APEX Growth Units per condition, including 12 for each experiment, and 3 spare units. Three distinct accessions of Brachypodium distachyon were used, including Bd21 (whose genome serves as a reference for Brachypodium), Bd21-3 (which has been optimized for use in transformation experiments), and Gaz-8 (which displays different root-growth behaviors relative to Bd21 and Bd21-3 when grown on the ground). Four chambers (biological repeats) per accession were tested under microgravity conditions on ISS, and another four were exposed to 1-g on the ground under conditions that mimicked the flight experiment (ground control).

This material was prepared for flight to the ISS on March 27-30, 2018, and launched on Space X-14 on April 2, 2018. Upon transferring the 12 seeded APEX growth chambers from stowage into the ISS, astronaut Scott Tingle activated the experiment by injecting growth medium into the units on April 12, 2018. Seeds were allowed to germinate in the presence of red light for 24 hours, then blue and green lights were turned on and the plants were allowed to grow for three more days. At the end of this growth period, astronaut Tingle collected each APEX growth unit, took photographs of the seedlings growing on each face of the foam block, harvested the seedlings and placed them into Kennedy Space Center Fixation Tubes (KFT) for fixation in the presence of RNAlater. For each APEX Growth Unit, a few seedlings displayed roots that grew away from the foam surface and showed evidence of stress. These seedlings were harvested separately from those whose root tips were still contacting the block surface at harvesting time. Fixation in RNAlater was carried out at room temperature for a period of 24 h. The KFTs were then transferred to a cold storage device (-80°C) and returned to the ground on the Dragon capsule.

The environmental conditions experienced by the plants during their growth in VEGGIE on ISS were recorded, and then recapitulated in plant growth units at KSC during a first ground-based control. At the end of this experiment, the seedlings were also fixed in RNAlater at room temperature for 24 hours, then frozen at -80°C. These ground-control samples were also returned to the Principal Investigator (PI) laboratory, as were the pictures of seedlings grown under both microgravity and 1-g conditions.

Microgravity-grown seedlings are morphologically altered compared to ground-controls The pictures of Brachypodium seedlings taken after 4 days of growth in the APEX Growth Units under microgravity and on the ground, were used to evaluate a potential effect of ISS-microgravity exposure on organs growth and seedling morphology. Seedlings of all three accessions displayed significantly shorter roots when grown under microgravity relative to ground controls. Furthermore, the shoots of microgravity-grown Bd21 seedlings were also shorter than ground controls, whereas those of Bd21-3 and Gaz-8 were similar under both conditions. We conclude that Brachypodium distachyon accessions display distinct adaptive responses to the microgravity environment encountered on ISS. In addition to accession-specific alterations of organs growth, we also observed that the primary roots of microgravity-grown seedlings displayed longer root hairs than those of ground-control samples, and this phenotype was observed for all three accessions tested. This phenotype is rather surprising as previous studies had shown an opposite effect of microgravity on Arabidopsis thaliana root hairs (4).

Brachypodium distachyon seedlings display organ-specific transcriptional responses to microgravity relative to ground control

To better understand the mechanisms that contribute to Brachypodium seedling adaptation to the microgravity environment of ISS, root and shoot tissues were dissected from frozen fixed Bd21 seedlings that had been exposed to the microgravity environment on ISS as well as ground-control materials. Total RNAs were extracted from these tissues and used to build TruSeq Stranded Total RNA libraries, which were sequenced in a Novaseq sequencer programmed to generate 150-bp paired-end reads. Library sequencing generated an average of 25 to 30 million reads per sample. Reads were trimmed to remove the adapter sequences using SKEWER, then filtered with FASTX_quality_filter to ensure that only high-quality reads (Phred scores above 35) are used for sequence alignments. The quality-filtered reads were mapped on the Brachypodium reference genome (version 3.1, from Phytozome) using the Bowtie2 algorithm. The TopHat 2 splice-junction mapper was used to compile transcript contigs, paying close attention to potential splice variants. HTseq-count was then used to define the numbers of reads per kilobase of transcript per million mapped reads (RPKM) for genes and isoforms in each analyzed sample. DESeq (1) was used to normalize the numbers of reads and identify significantly differentially expressed transcripts. EdgeR (9) was also used as an alternative method to identify differentially expressed genes, and genes identified as significantly differentially expressed by both packages were retained for further analysis. The results of our DESeq comparisons are presented below.

A principle component analysis (PCA) of expression profiles between experimental repeats revealed separate clustering between ground-control and microgravity-exposed samples, suggesting that there is more variation in expression profiles between environmental conditions than there is between repeats within a condition. Furthermore, ground-control root samples grouped more tightly in a sub-region of the PCA graph than those from microgravity-exposed root samples, suggesting a larger variation in expression profiles between repeats of the microgravity-exposed root samples relative to the ground controls.

Analysis of differential expression between microgravity-exposed and ground control seedlings identified 661 and 267 up-regulated genes in microgravity-exposed shoots and roots, respectively, relative to ground controls. Additionally, 1015 and 505 genes were found to be down-regulated in microgravity-exposed shoots and roots, respectively. A Gene Ontology (GO) annotation of these differentially expressed genes revealed an over-representation of genes potentially involved in plant responses to environmental stimuli, oxidative stress, reactive oxygen species and radiation, amongst other things.

Even though many genes ended up being differentially expressed between microgravity and ground control samples, most were organ-specific. This is consistent with similar observations made with Arabidopsis thaliana seedlings (5; 7). In fact, only 90 of the significantly differentially expressed genes are shared between roots and shoots. Of these, 35 are up-regulated and 30 down-regulated in both roots and shoots under microgravity. These genes constitute excellent candidates for a contribution to plant adaptive response to the microgravity environment of ISS. The remaining genes display opposite responses between shoots and roots. Several investigations of Arabidopsis seedling adaptation to spaceflight have also uncovered evidence of transcriptional responses involving genes that contribute to environmental and oxidative stress responses, as well as responses to reactive oxygen species (2-4; 6-8). On the other hand, our observation of enhanced expression of genes involved in radiation response under microgravity on ISS is particularly interesting considering the risks of exposure to cosmic radiations encountered by living organisms during spaceflight.

A limited number of Brachypodium distachyon genes found to respond to the microgravity environment of ISS also respond to GS and/or MS on the ground.

Because Brachypodium distachyon seedling-organ responses to GS had not been previously characterized, we carried out a second ground-control experiment in this project, aimed at identifying genes whose expression is significantly altered in response to GS and/or MS. We germinated and grew Brachypodium Bd21-accession seeds in APEX Growth Chambers, within a VEGGIE growth unit at KSC under conditions that were programmed to mimic those recorded on ISS during the spaceflight experiment. After 4 days of growth, the APEX growth units were either gravity-stimulated by 180° reorientation, mechano-stimulated by 360° rotation, or not stimulated (control), and the seedlings were allowed to grow in their new orientation for an additional 5 min. Each treatment involved four repeats (four APEX Growth units). After stimulation, the seedlings were harvested and fixed in RNAlater (7/19/2018), then frozen at -80°C after 1 day at room temperature. These samples were returned to the University of Wisconsin-Madison, and dissected to separate shoot and root tissues. RNA was extracted from each sample and analyzed by RNAseq to identify genes that are differentially expressed (activated or repressed) in response to GS or MS. Similar sequence-analysis packages were used as those described above for RNASeq analysis of Brachypodium organ responses to microgravity.

This analysis revealed only small numbers of significantly differentially expressed genes between GS-/MS-induced samples and ground controls. It is, however, particularly compelling to note that 10 genes were found to be differentially expressed in response to both GS and microgravity exposure in roots. Six of these genes were repressed upon GS and activated upon exposure to microgravity, whereas the other four genes were activated in response to GS and repressed by exposure to microgravity. Therefore, these 10 genes constitute strong candidates for a contribution to gravity response in roots. They will be subjected to functional characterization.

Equally interesting is another list of 10 genes that are transcriptionally responsive to both MS and microgravity exposure. Of these, eight were found to be down-regulated in response to MS and up-regulated upon exposure to microgravity, one was up-regulated in response to MS and down-regulated in response to microgravity, and the last one was found to be up-regulated in response to both treatments. One of these genes was also found in the list of 10 genes that are responsive to both GS and microgravity exposure. Overall, the latter group of 10 genes jointly regulated by MS and microgravity exposure are likely to contribute to plant responses to their mechanical environment. They will also be prioritized for further functional characterization.

In conclusion, we note that a vast majority of the genes found to be differentially expressed upon GS or MS on the ground are not transcriptionally responsive to the microgravity conditions experienced on ISS, suggesting that Brachypodium seedling organs interpret the microgravity environment of ISS very differently from GS and/or MS on the ground. This observation holds true for both shoot and root samples. It is compatible with similar observations made by others using Arabidopsis thaliana as a model (2-4; 6-8). A manuscript describing this work is in preparation.

Using a Genome-Wide Association Study approach to identify loci that contribute to root gravitropism and oscillatory root growth behavior in Brachypodium distachyon seedlings

In addition to the APEX-06 experiment described above, we have also completed our comparative analysis of root gravitropism and oscillatory root growth behavior on agar surfaces for 46 distinct Brachypodium distachyon accessions, using time lapse-analysis of root-growth behavior coupled with automated computer-driven biometric analysis of imaged roots. Our quantitative analysis of root growth behavior included determinations of root growth rates, root tip angles from the vertical, and elemental curvatures along the roots.

When growing on vertical plates, Brachypodium roots from most accessions grow straight down, as dictated by gravitropism. However, while doing so, the root-tip also tends to curve leftward and rightward of the vertical. The tip curvatures associated with these oscillations typically disappear as the differentially elongated cells migrate from the distal side of the elongation zone to more proximal regions, an auto-straightening process that is reminiscent of proprioception in animal systems. Upon 90-degree reorientation within the gravity field (gravity stimulation), the roots initiate a phase of rapid downward bending, which is followed by a second, slower phase of curvature response that is accompanied by root-tip oscillations similar to those preceding the gravity stimulus. Distinct Brachypodium accessions differ from each other in the frequency and amplitude of their root tip oscillations, as well as in the bending rate following gravity stimulation.

To better capture the genetic basis of variation between accessions for these traits, we developed a mathematical model that recapitulates the evolution of root tip angles from the vertical before, during and after gravity stimulation. When applied to the 46 accessions under investigation, this model is very effective at recapitulating accession-specific growth behaviors, with best-fit curves correlating well with the observed data (R-square larger than 0.9). These best-fit equations, along with Fast Fourier Transform analysis, allowed estimation of descriptive parameters for these behaviors, including root growth rate, rapid bending rates during phase 1 of gravitropism, and frequency and amplitude of oscillations.

Because each one of these 46 accessions had previously been subjected to genome sequencing, we were able to use genome-wide association studies (GWAS) to identify single nucleotide polymorphisms (SNPs) that are associated with these parameters. This analysis allowed us to identify linked loci whose contribution to oscillatory root growth behavior are being investigated using a combination of expression analysis, reverse genetics and cell biology approaches. A paper describing this research is also being prepared for publication.

Broader Impact of Our Work

Overall, these studies may impact our understanding of the molecular mechanisms that govern the regulation of coordinated cell expansion and its roles in growth regulation, plant morphogenesis and plant organs' growth responses and adaptation to gravity and/or microgravity. In the longer term, our studies may enable us to engineer plants that are better suited to function as key components of bioregenerative life support systems for long-term space exploration missions, or more simply for effective growth and productivity in natural and/or agricultural ecosystems on Earth.

New Technology Development: The foam-based vessel (APEX Growth Chamber) we developed in collaboration with Dr. Howard Levine and the KSC Flight Support Team has been submitted to NASA Technology Transfer System (KSC-14282).

Cited Literature

1. Anders S, Huber W. 2013. Differential expression of RNA-Seq data at the gene level - te DESeq package. ed. DV- Bioconductor

2. Basu P, Kruse C, Luesse D, Wyatt S. 2017. Growth in spaceflight hardware results in alterations to the transcriptome and proteome. Life Sci Space Res 15:88-96

3. Choi W, Barker R, Kim S, Swanson S, Gilroy S. 2019. Variation in the transcriptome of different ecotypes of Arabidopsis thaliana reveals signatures of oxidative stress in plant responses to spaceflight. Am J Bot 106:123-36

4. Kwon T, Sparks J, Nakashima J, Allen S, Tang Y, Blancaflor E. 2015. Transcriptional response of Arabidopsis seedlings during spaceflight reveals peroxidase and cell wall remodeling genes associated with root hair development. Am J Bot 102:21-35

5. Paul A, Daugherty C, Bihn E, Chapman D, Norwood K, Ferl R. 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis. Plant Physiol 126:613-21

6. Paul A, Sng N, Zupanska A, Krishnamurthy A, Schultz E, Ferl R. 2017. Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight? PLoS One 12:e0180186

7. Paul A, Zupanska A, Schultz E, Ferl R. 2013. Organ-specific remodeling of the Arabidopsis transcriptome in response to spaceflight. BMC Plant Biol 13:112

8. Paul A-L, Zupanska A, Ostrow D, Zhang Y, Sun Y, et al. 2012. Spaceflight transcriptomes: Unique responses to a novel environment. Astrobiology 12:40-56

9. Robinson M, McCarthy D, Smyth G. 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139-40

JUNE 2019 REPORT: As summarized in our previous progress reports, we developed a novel plant growth unit (named APEX growth chamber) to support the growth of Brachypodium distachyon seedlings under microgravity in the International Space Station (ISS). This system is based on an Oasis foam-based growth device previously used by Dr. Howard Levine to grow plants in space (NASA Space Biology Program). Our units were designed in a collaborative effort involving Dr. Levine and the NASA Flight Support Team. With this system, seeds are inserted pre-flight into a dry block of foam, which is mounted on a medium injection device within a Magenta box. The seeds are kept in a dormant dry state, unable to germinate during spaceflight to the ISS. When the experiment is ready for initiation on ISS, an astronaut can easily inject liquid growth medium into the foam support through the injection device, thereby imbibing the seeds and triggering their germination under red light.

The flight experiment and corresponding ground control were carried out in March 2018 (APEX-06 mission). These experiments involved a total of 15 APEX Growth Units per condition, including 12 for each experiment and 3 spare ones. Three distinct accessions of Brachypodium distachyon were used, including Bd21 (whose genome serves as a reference for Brachypodium), BD21-3 (which has been optimized for use in transformation experiments), and Gaz-8 (which displays different root growth behaviors relative to Bd21 and Bd21-3 when grown on the ground). Four chambers (biological repeats) per accession were tested under microgravity conditions on ISS, and another four were exposed to 1-g on the ground under conditions that mimicked the flight experiment (ground control).

As previously reported, this material was prepared for flight to the ISS on March 27-30, 2018, and launched on SpaceX-14 on April 2, 2018. Upon transferring the 12 seeded APEX growth chambers from stowage into the ISS, astronaut Scott Tingle activated the experiment by injecting growth medium into the units on April 12, 2018. Seeds were allowed to germinate in the presence of red light for 24 hours, then blue and green lights were turned on and the plants were allowed to grow for three more days. At the end of this growth period, astronaut Tingle collected each APEX growth unit, took photographs of each face, harvested the seedlings, and placed them into KFT for fixation in the presence of RNAlater. For each APEX Growth Unit, a few seedlings displayed roots that grew away from the foam surface and showed evidence of stress. These seedlings were harvested separately from those whose root tips were still contacting the block surface at harvesting time. Fixation in RNAlater was carried out at room temperature for a period of 24 h. The KFTs were then transferred to a cold storage device (-80°C) and returned to the ground on the Dragon capsule.

The environmental conditions experienced by the plants during their growth period in VEGGIE on ISS were recorded, and then recapitulated in plant growth units at KSC during a first ground-based control. At the end of this experiment, the seedlings were also fixed in RNAlater. After similar storage under -80°C conditions, the samples were returned to the Principal Investigator (PI) laboratory, as were the pictures of seedlings grown under both microgravity and 1-g conditions. As reported last year, seedlings grown under microgravity displayed significant root-growth inhibition compared to ground controls for all accessions tested. The shoots of microgravity-grown Bd21 seedlings were also shorter than ground control, but those of Bd21-3 and Gaz-8 were similar under both gravity conditions. We conclude that distinct Brachypodium distachyon accessions display distinct adaptive responses to the microgravity environment encountered on ISS. In addition to accession-specific slight alterations of organs growth, we also observed that the primary roots of microgravity-grown seedlings displayed longer root hairs than those of ground-control samples.

To better understand the mechanisms that contribute to Brachypodium seedlings adaptation to the microgravity environment of ISS, root and shoot tissues were dissected from frozen fixed Bd21 seedlings that had been exposed to the microgravity environment on ISS as well as ground-control material. Total RNAs were extracted from these tissues and used to build TruSeq Stranded Total RNA libraries, which were sequenced in a Novaseq sequencer programmed to generate 150-bp paired-end reads. Library sequencing generated an average of 25 to 30 million reads per sample. Sequence analysis involved a pipeline that included FastQC to control for the quality of sequence reads and filter out poor reads, Tophat2 to align each reads to the reference Brachypodium genome, Bowtie2 to build an index, HTseq to count the number of reads assigned to each annotated gene/transcript, and DEseq (R package) to evaluate differential expression between microgravity-exposed and ground control samples.

A principle component analysis (PCA) of expression profiles between experimental repeats revealed separate clustering between ground-control and microgravity-exposed samples, suggesting that there is more variation in expression profiles between environmental conditions than there is between repeats within a condition. Furthermore, ground-control root samples grouped more tightly in a sub-region of the PCA graph than those from microgravity-exposed root samples, suggesting a larger variation in expression profiles exists between repeats of the microgravity-exposed root samples relative to the ground controls.

Initial analysis of differential expression between microgravity-exposed and ground control seedlings identified 398 and 159 significantly up-regulated genes in microgravity-exposed shoots and roots, respectively, relative to ground controls. Additionally, 867 and 380 significantly down-regulated genes were found in microgravity-exposed shoots and roots, respectively. A Gene Ontology (GO) annotation of these differentially expressed genes revealed an over-representation of genes potentially involved in plant responses to environmental stimuli and oxidative stress, amongst other things.

Because the Brachypodium distachyon seedling organ responses to gravity stimulation have not been well characterized, we also carried out a second control experiment in this project, aimed at identifying genes whose expression is significantly altered in response to gravity- and/or mechano-stimulation. As reported last year, we germinated and grew Bd21 seeds in APEX Growth Chambers, within a VEGGIE unit, under growth conditions that were programmed to mimic those recorded on ISS during the spaceflight experiment. After four days of growth, the APEX growth units were either gravity-stimulated by 180° reorientation, mechano-stimulated by 360° rotation, or unstimulated (control), and the seedlings were allowed to grow in their new position for five minutes. Each stimulus involved four repeats (four APEX Growth units). After 5-min incubations, the seedlings were harvested and fixed in RNAlater (7/19/2018), then frozen at -80°C after one day of fixation. These samples were returned to the University of Wisconsin-Madison where each sample was dissected to separate shoot and root tissues. RNA was extracted from each sample and analyzed by RNAseq to identify genes that are differentially expressed between treatments. Currently, the analytical pipeline described above is being used to analyze the RNAseq profiles and identify genes that are significantly differentially expressed (activated or repressed) in response to mechano- or gravity-stimulation. The groups of genes found to be differentially expressed under microgravity on ISS will be compared to those that are differentially expressed in response to gravity- or mechano-stimulation under 1-g. Reverse genetics will then be used to investigate the contribution of these genes to plant responses to microgravity, gravity- or mechano-stimulation.

In addition to the APEX-06 experiment described above, we have also completed our comparative analysis of root gravitropism and oscillatory root growth behavior on agar surfaces for 45 distinct Brachypodium distachyon accessions, using time lapse-analysis of root growth behavior coupled with automated computer-driven biometric analysis of imaged roots. Our quantitative analysis of root growth behavior included determinations of root growth rates, root tip angles from the vertical, and elemental curvatures along the roots.

When growing on vertical plates, Brachypodium roots from most accessions grow straight down, as dictated by gravitropism. However, while doing so, the root-tip also tends to curve leftward and rightward of the vertical. The tip curvatures associated with these oscillations typically disappear as the differentially elongated cells migrate from the distal side of the elongation zone to more proximal regions, an auto-straightening process that is reminiscent of proprioception in animal systems. Upon 90-degree reorientation within the gravity field (gravity stimulation), the roots initiate a phase of rapid downward bending, which is followed by a second, slower phase of curvature response that is accompanied by root-tip oscillations similar to those preceding the gravity stimulus. Distinct Brachypodium accessions differ from each other in the frequency and amplitude of their root tip oscillations, as well as in the bending rate following gravity stimulation.

To better capture the genetic basis of variation between accessions for these traits, we developed a mathematical model that recapitulates the evolution of root tip angles from the vertical before, during, and after gravity stimulation. When applied to the 45 accessions under investigation, this model was very effective at recapitulating accession-specific growth behaviors, with best-fit curves correlating well with the observed data (R-square larger than 0.9). These best-fit equations, along with Fast Fourier Transform analysis, allowed estimation of descriptive parameters for these behaviors, including root growth rate, rapid bending rates during phase 1 of gravitropism, and frequency and amplitude of oscillations.

Because each one of these 45 accessions had previously been subjected to genome sequencing, we were able to use genome-wide association studies (GWAS) to identify single nucleotide polymorphisms (SNPs) that are associated with these parameters. SNPs showing significant probabilities of association with several growth parameters including the maximal rate of rapid bending and the amplitude of oscillations, have been identified and are being characterized. Overall, these studies have allowed us to identify linked loci whose contribution to oscillatory root growth behavior are being investigated using a combination of expression analysis, reverse genetics and cell biology approaches.

Overall, these studies may impact our understanding of the molecular mechanisms that govern the regulation of coordinated cell expansion and its roles in growth regulation, plant morphogenesis, and plant organs' growth responses and adaptation to gravity and/or microgravity. In the longer term, our studies may enable us to engineer plants that are better suited to function as key components of bioregenerative life support systems for long-term space exploration missions, or more simply for effective growth and productivity in natural and/or agricultural ecosystems on Earth.

NOTE: End date changed to 09/15/2019 per NSSC information (Ed., 3/12/19)

NOTE: End date changed to 3/11/2019 per NSSC information (Ed., 9/14/18)

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

NOTE: End date changed to 9/11/2017 per NSSC information (Ed., 6/14/16)

This year, we tested and refined this novel experimental protocol, which successfully passed a Science Verification Test (SVT) at Kennedy Space Center (fall of 2017), and an Experiment Verification Test (EVT) in January of 2018. This experiment uses three Brachypodium distachyon accessions known to display distinct root growth behaviors under normal gravity conditions: Bd21 (whose genome has served as a reference for Brachypodium), BD21-3 (which has been optimized for use in transformation experiments), and Gaz-8 (which displays different root growth behaviors relative to Bd21 and Bd21-3 when grown on the ground).

These tests successfully demonstrated high germination rates for all three accessions, sufficient seedling organ growth to allow subsequent molecular characterization of expression profiles, and ability to extract good quality RNA for subsequent to RT-qPCR analysis. Therefore, this project was given the green light to proceed to its implementation phase on ISS, under the name 'APEX-06'. The material was prepared for flight on March 27-30, 2018, and launched on SpaceX-14 on April 2, 2018.

One astronaut activated the experiment on ISS by injecting growth medium into the 12 APEX growth chambers (4 repeats for each of the three accessions described above) on April 12, 2018. Seeds were allowed to germinate in the presence of red light for 24 hours, then blue and green light were added for an additional 3 days. At the end of this growth period, astronaut Dr. Tingle recovered the APEX growth chambers from VEGGIE, took photographs of the developing seedlings on each face of the foam block, harvested the seedlings, and placed them into Kennedy Fixation Tubes (KFT) for fixation with RNA later. Most APEX Growth Chambers contained a few seedlings that displayed an unusual growth pattern with roots detached from the foam surface, therefore showing evidence of stress. These seedlings were harvested separately from those whose root tips were still contacting the block surface. Fixation was allowed to proceed for a period of 24 h, at which time the KFTs were transferred to cold storage (-80°C) and returned to the ground for molecular analysis in the Principal Investigator's laboratory.

A parallel time-delayed ground-based control experiment was carried out at KSC, during which the environmental conditions experienced by the plants during their growth period in VEGGIE on ISS were recapitulated in plant growth units at KSC. At the end of this experiment, the seedlings were also photographed, harvested, and fixed in RNA later for subsequent RNA extraction and transcriptomic analysis.

Photographs taken during both the flight experiment on ISS and the ground-based control experiment at KSC were used to investigate seedling organs growth under microgravity or control 1 g conditions, revealing a slight alteration in the rate of growth under microgravity.

To better understand the molecular mechanisms that underlie Brachypodium seedling organs adaptation to the microgravity environment, fixed frozen seedlings will be dissected to separate shoots and roots. RNA will be extracted from these tissues and used in RNA-seq experiments aimed at comparing organ-specific expression profiles between microgravity-grown and control samples.

A second ground-based control experiment was also carried out at KSC by Dr. Jeffrey Richards in July 2018, to investigate the molecular mechanisms that modulate root and shoot responses to gravistimulation and mechanostimulation under 1g conditions. In this experiment, Bd21 seedlings were germinated and grown in APEX growth chambers under the same conditions as those experienced during the APEX-06 mission on ISS. After 4 days of growth, the APEX growth units were either gravistimulated by a 180° reorientation, mechanostimulated by a 360° stimulus, or unstimulated (control). Four APEX growth chambers were subjected to each one of these treatments (four biological repeats). After 5 min of stimulation, the seedlings were harvested from the chambers and fixed in RNA later, then frozen at -80°C. Fixed seedlings will be dissected to separate shoots from roots. RNA will then be extracted from these tissues, and used in an RNAseq analysis to characterize their transcriptomic profiles. Groups of genes found to be differentially expressed under microgravity in ISS relative to the first ground-based control will be compared to those found to be differentially expressed in response to gravistimulation under 1 g or in response to mechanostimulation (as determined in the second ground-based control). Groups of genes that are found to be differentially expressed under both microgravity and gravistimulation conditions will be retained for functional analysis as they are more likely to directly contribute to gravity signal transduction.

In addition to the APEX-06 experiment, we have also continued our analysis of Brachypodium root gravitropism and oscillatory root growth behavior on agar surfaces. Last year, we reported using the natural variation that exists between 42 Brachypodium accessions to investigate the molecular mechanisms that underlie the variation in kinetics of root gravitropism and properties of oscillatory root growth. This year, we have extended this analysis to include more Brachypodium accessions and a larger number of seedlings per accession, making the identification of associated loci by Genome-Wide Association Study (GWAS) more robust and meaningful. Importantly, this analysis allowed the identification of several polymorphisms associated with quantitative parameters that recapitulate different aspects of root-tip oscillatory growth. Some of the significant SNPs identified in this work mapped to genes that encode a sugar-binding protein kinase and a polar auxin transporter. These genes' annotations suggest roles in proprioception and auxin transport/signaling, respectively, and experiments are underway to verify their contribution to the control of oscillatory root growth.

Overall, these studies should improve our understanding of the molecular mechanisms that control coordinated cell expansion in roots and its roles in growth regulation, morphogenesis, and growth responses to gravity and/or microgravity. These studies may impact our ability to engineer plants that are better suited for use in the bioregenerative life-support systems that will accompany long-term space exploration missions, or more simply for effective growth and productivity in natural and/or agricultural ecosystems on Earth.

NOTE: End date changed to 9/11/2017 per NSSC information (Ed., 6/14/16)

To be fully informative, the planned experiments will require comparing the morphology and transcriptomic profiles of Brachypodium seedlings exposed to either microgravity or 1g during their entire growth period, from germination to harvesting. This implies that the microgravity-exposed plants will have to derive from seeds that germinated after transfer into VEGGIE on ISS, under microgravity, or on Earth in the ground control. As reported last year, we tested two experimental approaches allowing to plate seeds on growth substrate on the ground, before flight, and transporting the planted material to ISS for germination and growth in VEGGIE. In the first protocol, Brachypodium seeds are plated on agar-containing medium within Petri dishes. The seeds are prevented from germinating prematurely by continuous blue-light treatment in the cold (4oC). Upon transfer into VEGGIE on ISS, germination can be induced by exposing the dishes to red light at room temperature for 24-36 h. Unfortunately, this system was deemed difficult to implement during spaceflight because it would require a significant power source to light up the plated seeds and keep the environmental temperature at 4oC during flight.

Considering this limitation, an alternative protocol was developed under advice from Dr. Howard Levine, with technical assistance from the Kennedy Space Center Support Team. In this protocol, up to 24 surface-sterilized Brachypodium seeds are embedded in a block of dry Oasis Foam contained within a Magenta Jar. Because both foam and seeds are dry, the plant material remains dormant during vehicle takeoff, travel to ISS, and docking. After transfer into VEGGIE, astronauts will activate seed germination by injecting liquid medium into the foam and exposing the jars to red light for 24-36 hours. Subsequently, green and blue LED lights will be turned on, and the seedlings will be allowed to grow for a total of 5 days. The seedlings will then be photographed, harvested, transferred into Kennedy Fixation Tubes (KFTs), and fixed in RNALater for 24-36 h at room temperature. The fixed material will then be frozen at -70oC and stored until return to the ground.

This year, we have demonstrated the effectiveness of this medium-injection system to trigger germination. We also designed a new medium injection device that fits within a modified Magenta Jar, and allows effective injection of medium into the foam without opening the jar. This system is designed to include a connecting spout that protrudes through a hole at the bottom of the Magenta jar. This spout allows easy snap-in connection to a hose attached to a growth medium-filled syringe. With this system it should be easier for the astronauts to connect a medium-filled syringe to the Magenta Jar spout and inject a defined amount of liquid into the foam, using the syringe. To allow more homogenous distribution of liquid within the foam, a layer of gauze is sawed on the surface of the foam, itself surrounded by a layer of Nitex membrane that prevents roots from penetrating the foam block. Seeds are embedded into the foam through slits cut within the Nitex and gauze surfaces. Seeds are stabilized within the slits by a drop of guar gum, which prevents them from dispersal by the vibrations typically encountered during vehicle takeoff. An adapter platform will also be designed to anchor up to 12 Magenta jars within VEGGIE (four biological repeats for each one of the three accessions: Bd21, Bd21-3, and Gaz-8).

A Science Verification Test (SVT) was carried out at Kennedy Space Center on June 12-14, 2017. After seed planting into the dry foam and medium injection, the Magenta jars were transferred into a VEGGIE unit placed in a reach-in growth chamber programmed to reproduce the temperature, humidity, and CO¬2 profiles encountered on ISS. At the end of the growth period, Brachypodium seedlings were photographed, harvested, and fixed in RNA later within KFTs. They were frozen, and then returned to the Principal Investigator (PI) laboratory for molecular analysis. Frozen fixed seedlings were dissected, generating separate shoot and root samples that were processed for RNA extraction. These extracted RNAs are currently being analyzed for quality and ability to serve as effective substrates for RT-PCR analyses. In parallel, the pictures of seedlings grown in the Magenta Growth Chamber (MGC) are being analyzed to determine germination rate and organs growth.

In addition to preparing for the APEX-06 spaceflight experiment, we have also continued our analysis of Brachypodium root gravitropism and growth behavior on agar surfaces. Last year, we reported on the development of time-lapse video imaging of Brachypodium root growth to evaluate the kinetics of root gravitropism and quantify the oscillatory growth behavior displayed by the roots of 42 distinct accessions developing on agar-based media. This analysis allowed us to identify several candidate loci that were associated with quantitative traits relevant to gravitropism and oscillatory root growth. This year, we have continued these analyses, developing a more robust imaging platform for automated analysis of seedling root growth behavior, and repeating our analysis on a larger number of seedlings for each accession. We have also acquired 60 additional Brachypodium accessions with fully sequenced genomes (kindly provided to us by Dr. John Vogel, University of California-Berkeley), which we are propagating under our own growth conditions for subsequent genome-wide association studies (GWAS) analysis. Furthermore, we initiated an analysis of primary root growth in the Bd21 accession, with the objective of mapping the meristem and elongation zones along the root tip for this accession, and also evaluating the relative contribution of each root-tip zone to the gravitropic response as well as the curvatures associated with oscillatory growth and autostraightening. A morphometric analysis of cell sizes along the root tip along with an analysis of the spatial profile of velocities using the Stripflow software (developed by Dr. Tobias Baskin, University of Massachusetts, Amherst, MA), are underway to evaluate the spatio-temporal distribution of curvatures along the root tip, hence identify key growth processes that are targeted by the gravity stimuli and/or the autostraightening response. Furthermore, transgenic lines expressing auxin-level and auxin-activity reporters, along with several reporters of auxin efflux facilitator localization (all kindly provided to us by Dr. Devin O'Connor, University of Cambridge, UK) are being used to evaluate the potential contribution of auxin redistribution and response to the various phases of root gravitropism and root oscillatory growth. Together, this combination of physiological, genetic, and cell biological investigations will provide new insights into the molecular mechanisms that modulate root gravitropism and oscillatory growth in monocots in general, and in Brachypodium distachyon in particular. In the longer term, these studies will yield novel information that may facilitate the development of novel breeding strategies or cultivation approaches to improve crop productivity on Earth and during spaceflight, considering the relevance of root growth behavior and root system architecture to plant productivity and survival under challenging conditions.

The following publication is in production:

Books/Book Chapters: Gibbs, N.M., Vaughn Rouhana, L., and Masson, P.H. "Quantitative Trait Loci for Root Growth Response to Cadaverine in Arabidopsis. " in "Plant Polyamines: Methods and Protocols." Ed. Alcazar, R., and Tiburcio, A. Springer, expected publication Dec-2017

Last year, we reported having optimized the growth conditions for Brachypodium on agar-based media in Petri dishes, working out protocols to inhibit germination for a sufficiently long period of time to allow seeds plated on the ground to germinate only after reaching the microgravity environment of the ISS. This protocol included treating plated seeds with blue light in the cold (4 degrees C) to inhibit germination for up to 12 days, followed by a red light treatment at 22 degrees C to promote synchronized germination. We further optimized this treatment such that seeds from all four chosen accessions would respond appropriately. Unfortunately, it recently became apparent that the equipment needed to implement this protocol during spaceflight, including a power source to drive continuous LED blue light and a sufficiently powerful cooling device to dissipate light-derived heat and maintain a temperature of 4 degrees C, would likely be prohibitive and/or difficult to access.

Considering these difficulties, we restructured our research project. Under excellent guidance and advice from Dr. Howard G. Levine (Kennedy Space Center (KSC), Florida) and members of the KSC technical-support team, we developed a novel seedling culturing system for Brachypodium distachyon, which relies on the use of Oasis Horticubes as growth substrate. This foam-like material can be carved to fit within either a Petri dish or a Magenta box. Seeds are embedded within the dry foam, where they remain dormant until the foam is hydrated by injection of liquid 1/2 MS medium. Germination is then synchronized with a 24-h red light treatment at 22 degrees C. Under these conditions, the seeds germinate quickly and synchronously, and the corresponding seedlings grow at faster rate than on agar-based medium. The advantage of this system is that germination will not occur unless triggered by wetting the foam. Therefore, there is no need to use blue light in the cold to inhibit germination during take off and travel to ISS. This revised protocol works very well for all Brachypodium accessions chosen in our investigations, and will substantially simplify the entire spaceflight protocol.

One limitation of this system is the need to physically inject liquid medium into the foam to trigger germination. In the microgravity environment of ISS, this process will have to be carried out by the astronauts under conditions that cannot be fully sterile if the plates have to be opened. To decrease the amount of time spent by the ISS crew to inject medium into the foam and increase the likelihood that the material will remain sterile during injection, we are designing a foam-support system that carries a separate, sealed liquid-medium tank. A simple lever system reachable from the outside of the dish, will be used to inject the medium into the foam. These levers will have to be accessible from the outside of the dish to allow easy access by the astronauts without compromising sterility. Because the walls of Petri dishes are too fragile to allow such redesign, we will use Magenta boxes in our experimental set up, which can be modified more effectively without affecting material sturdiness.

In addition to preparing for the spaceflight experiment, we have also continued our analysis of Brachypodium root gravitropism and growth behavior on agar surfaces. The kinetics of root gravitropism and oscillatory growth behavior displayed by most available Brachypodium accessions were carefully analyzed by time-lapse microscopy, and a mathematical model was developed that recapitulates this behavior. Data fitting of the model allowed us to quantify various parameters that represent the behaviors of 42 distinct accessions, including time of maximum gravicurvature rate during a graviresponse, angle of transition between rapid and slower phases of gravicurvature, and frequency and amplitude of root-tip oscillations. These calculated parameters were used in genome-wide association studies (GWAS) to identify loci that contribute to the variation between accessions. Strong candidate associations were observed for the time to maximum gravitropic bending rate and for the amplitude of oscillations. Associated loci are being functionally analyzed using reverse genetics.

These studies will provide new insights into the molecular mechanisms that modulate root gravitropism in monocots in general, and in Brachypodium distachyon in particular. They will also elucidate some of the processes that underlie the oscillatory behavior displayed by growing monocot root tips. Considering the importance of root growth behavior and architecture in plant productivity and fitness, this information may be useful in breeding programs aimed at improving crop productivity both on Earth and during spaceflight, at least in the longer term.

During this first year of our project, we have been able to better define the experimental parameters that will be needed to grow sterile Brachypodium distachyon seedlings in Petri dishes in the microgravity environment of ISS, within the VEGGIE chamber, for morphological and molecular comparisons with ground-based controls. A simple method for surface-sterilization of the seeds has been developed, which prevents microbial contamination of the cultures while maintaining full germination capacity. Medium composition and seed plating have been optimized to assure maintenance of medium integrity and prevent seeds from bouncing off the surface during spacecraft takeoff. We also demonstrated that application of blue-light during cold storage inhibits the germination of plated seeds for a period long enough to allow flight preparations, plates loading on the spacecraft, vehicle takeoff, travel to and docking with ISS, and material transfer to the growth chamber on ISS. Furthermore, a red-light treatment was developed to promote synchronized seed germination after removal from cold storage and transfer to the growth chamber. We also established the maximal period of seedling growth allowed considering the space constraints imposed by the dishes, and are in the process of optimizing protocols for seedling fixation at the end of the experiment, and RNA extraction for subsequent transcription profiling.

These methods have been optimized for Bd21-3, an easily transformable and widely used Brachypodium accession. However, in the next few weeks, we will also test them on five other accessions, which we plan to incorporate in the microgravity-adaptation experiment because they display distinct root sensitivities to gravity and/or show distinct growth behaviors in response to gravistimulation on Earth. For all six accessions, plants have been propagated, generating more than enough seeds to carry out both microgravity and ground-based control experiments. We expect being able to complete a Science Verification Test at Kennedy Space Center (KSC) in December of 2015, as recently discussed with the KSC Support Team.

In addition to this Definition phase of our project, we have developed more sophisticated approaches to analyze the gravisensitivities of our Brachypodium accessions, and have discovered a novel root-growth behavior associated with long-term clinorotation, which we hope to correlate with gravisensitivity. We have also collaborated with Dr. Nathan Miller in the Spalding laboratory (University of Wisconsin-Madison, Botany Department) to develop a high-definition image acquisition and analysis platform that allows extraction of defining spatio-temporal characteristics of Brachypodium root graviresponses. This analytical platform will be adopted to analyze all available Brachypodium accessions, and mathematical models will be fit to the data as a way to extract informative parameters defining each accession's gravi- and proprio-sensitivity. These parameters will then be introduced into Genome-Wide Association Study (GWAS) programs to identify novel loci that contribute to gravisensing and proprioception in this system.