Epigenomics in an extraterrestrial environment: organ-specific alteration of DNA methylation and gene expression elicited by spaceflight in Arabidopsis thaliana.

BACKGROUND: Plants adapted to diverse environments on Earth throughout their evolutionary history, and developed mechanisms to thrive in a variety of terrestrial habitats. When plants are grown in the novel environment of spaceflight aboard the International Space Station (ISS), an environment completely outside their evolutionary history, they respond with unique alterations to their gene expression profile. Identifying the genes important for physiological adaptation to spaceflight and dissecting the biological processes and pathways engaged by plants during spaceflight has helped reveal spaceflight adaptation, and has furthered understanding of terrestrial growth processes. However, the underlying regulatory mechanisms responsible for these changes in gene expression patterns are just beginning to be explored. Epigenetic modifications, such as DNA methylation at position five in cytosine, has been shown to play a role in the physiological adaptation to adverse terrestrial environments, and may play a role in spaceflight as well. RESULTS: Whole Genome Bisulfite Sequencing of DNA of Arabidopsis grown on the ISS from seed revealed organ-specific patterns of differential methylation compared to ground controls. The overall levels of methylation in CG, CHG, and CHH contexts were similar between flight and ground DNA, however, thousands of specifically differentially methylated cytosines were discovered, and there were clear organ-specific differences in methylation patterns. Spaceflight leaves had higher methylation levels in CHG and CHH contexts within protein-coding genes in spaceflight; about a fifth of the leaf genes were also differentially regulated in spaceflight, almost half of which were associated with reactive oxygen signaling. CONCLUSIONS: The physiological adaptation of plants to spaceflight is likely nuanced by epigenomic modification. This is the first examination of differential genomic methylation from plants grown completely in the spaceflight environment of the ISS in plant growth hardware developed for informing exploration life support strategies. Yet even in this optimized plant habitat, plants respond as if stressed. These data suggest that gene expression associated with physiological adaptation to spaceflight is regulated in part by methylation strategies similar to those engaged with familiar terrestrial stress responses. The differential methylation maps generated here provide a useful reference for elucidating the layers of regulation of spaceflight responses.

Skewing in Arabidopsis roots involves disparate environmental signaling pathways.

BACKGROUND: Skewing root patterns provide key insights into root growth strategies and mechanisms that produce root architectures. Roots exhibit skewing and waving when grown on a tilted, impenetrable surface. The genetics guiding these morphologies have been examined, revealing that some Arabidopsis ecotypes skew and wave (e.g. WS), while others skew insignificantly but still wave (e.g. Col-0). The underlying molecular mechanisms of skewing and waving remain unclear. In this study, transcriptome data were derived from two Arabidopsis ecotypes, WS and Col-0, under three tilted growth conditions in order to identify candidate genes involved in skewing. RESULTS: This work identifies a number of genes that are likely involved in skewing, using growth conditions that differentially affect skewing and waving. Comparing the gene expression profiles of WS and Col-0 in different tilted growth conditions identified 11 candidate genes as potentially involved in the control of skewing. These 11 genes are involved in several different cellular processes, including sugar transport, salt signaling, cell wall organization, and hormone signaling. CONCLUSIONS: This study identified 11 genes whose change in expression level is associated with root skewing behavior. These genes are involved in signaling and perception, rather than the physical restructuring of root. Future work is needed to elucidate the potential role of these candidate genes during root skewing.

CARM1 is required for proper control of proliferation and differentiation of pulmonary epithelial cells.

Coactivator-associated arginine methyltransferase I (CARM1; PRMT4) regulates gene expression by multiple mechanisms including methylation of histones and coactivation of steroid receptor transcription. Mice lacking CARM1 are small, fail to breathe and die shortly after birth, demonstrating the crucial role of CARM1 in development. In adults, CARM1 is overexpressed in human grade-III breast tumors and prostate adenocarcinomas, and knockdown of CARM1 inhibits proliferation of breast and prostate cancer cell lines. Based on these observations, we hypothesized that loss of CARM1 in mouse embryos would inhibit pulmonary cell proliferation, resulting in respiratory distress. By contrast, we report here that loss of CARM1 results in hyperproliferation of pulmonary epithelial cells during embryonic development. The lungs of newborn mice lacking CARM1 have substantially reduced airspace compared with their wild-type littermates. In the absence of CARM1, alveolar type II cells show increased proliferation. Electron microscopic analyses demonstrate that lungs from mice lacking CARM1 have immature alveolar type II cells and an absence of alveolar type I cells. Gene expression analysis reveals a dysregulation of cell cycle genes and markers of differentiation in the Carm1 knockout lung. Furthermore, there is an overlap in gene expression in the Carm1 knockout and the glucocorticoid receptor knockout lung, suggesting that hyperproliferation and lack of maturation of the alveolar cells are at least in part caused by attenuation of glucocorticoid-mediated signaling. These results demonstrate for the first time that CARM1 inhibits pulmonary cell proliferation and is required for proper differentiation of alveolar cells.

Root Skewing-Associated Genes Impact the Spaceflight Response of Arabidopsis thaliana.

The observation that plant roots skew in microgravity recently refuted the long-held conviction that skewing was a gravity-dependent phenomenon. Further, spaceflight root skewing suggests that specific root morphologies and cell wall remodeling systems may be important aspects of spaceflight physiological adaptation. However, connections between skewing, cell wall modification and spaceflight physiology are currently based on inferences rather than direct tests. Therefore, the Advanced Plant Experiments-03-2 (APEX-03-2) spaceflight study was designed to elucidate the contribution of two skewing- and cell wall-associated genes in Arabidopsis to root behavior and gene expression patterns in spaceflight, to assess whether interruptions of different skewing pathways affect the overall spaceflight-associated process. SPIRAL1 is a skewing-related protein implicated in directional cell expansion, and functions by regulating cortical microtubule dynamics. SKU5 is skewing-related glycosylphosphatidylinositol-anchored protein of the plasma membrane and cell wall implicated in stress response signaling. These two genes function in different cellular pathways that affect skewing on the Earth, and enable a test of the relevance of skewing pathways to spaceflight physiological adaptation. In this study, both sku5 and spr1 mutants showed different skewing behavior and markedly different patterns of gene expression in the spaceflight environment. The spr1 mutant showed fewer differentially expressed genes than its Col-0 wild-type, whereas sku5 showed considerably more than its WS wild-type. Developmental age played a substantial role in spaceflight acclimation in all genotypes, but particularly in sku5 plants, where spaceflight 4d seedlings had almost 10-times as many highly differentially expressed genes as the 8d seedlings. These differences demonstrated that the two skewing pathways represented by SKU5 and SPR1 have unique and opposite contributions to physiological adaptation to spaceflight. The spr1 response is less intense than wild type, suggesting that the loss of SPR1 positively impacts spaceflight adaptation. Conversely, the intensity of the sku5 responses suggests that the loss of SKU5 initiates a much more complex, deeper and more stress related response to spaceflight. This suggests that proper SKU5 function is important to spaceflight adaptation.

A member of the CONSTANS-Like protein family is a putative regulator of reactive oxygen species homeostasis and spaceflight physiological adaptation.

A feature of the physiological adaptation to spaceflight in Arabidopsis thaliana (Arabidopsis) is the induction of reactive oxygen species (ROS)-associated gene expression. The patterns of ROS-associated gene expression vary among Arabidopsis ecotypes, and the role of ROS signalling in spaceflight acclimation is unknown. What could differences in ROS gene regulation between ecotypes on orbit reveal about physiological adaptation to novel environments? Analyses of ecotype-dependent responses to spaceflight resulted in the elucidation of a previously uncharacterized gene (OMG1) as being ROS-associated. The OMG1 5' flanking region is an active promoter in cells where ROS activity is commonly observed, such as in pollen tubes, root hairs, and in other tissues upon wounding. qRT-PCR analyses revealed that upon wounding on Earth, OMG1 is an apparent transcriptional regulator of MYB77 and GRX480, which are associated with the ROS pathway. Fluorescence-based ROS assays show that OMG1 affects ROS production. Phylogenetic analysis of OMG1 and closely related homologs suggests that OMG1 is a distant, unrecognized member of the CONSTANS-Like protein family, a member that arose via gene duplication early in the angiosperm lineage and subsequently lost its first DNA-binding B-box1 domain. These data illustrate that members of the rapidly evolving COL protein family play a role in regulating ROS pathway functions, and their differential regulation on orbit suggests a role for ROS signalling in spaceflight physiological adaptation.

Phenotypic characterization of an Arabidopsis T-DNA insertion line SALK_063500.

In this article we report the identification of a homozygous lethal T-DNA (transfer DNA) line within the coding region of the At1G05290 gene in the genome of Arabidopsis thaliana (Arabidopsis) line, SALK_063500. The T-DNA insertion is found within exon one of the AT1G05290 gene, however a homozygous T-DNA allele is unattainable. In the heterozygous T-DNA allele the expression levels of AT1G05290 were compared to wild type Arabidopsis (Col-0, Columbia). Further analyses revealed an aberrant silique phenotype found in the heterozygous SALK_063500 plants that is attributed to the reduced rate of pollen tube germination. These data are original and have not been published elsewhere.

Utilization of single-image normalized difference vegetation index (SI-NDVI) for early plant stress detection.

PREMISE OF THE STUDY: An imaging system was refined to monitor the health of vegetation grown in controlled conditions using spectral reflectance patterns. To measure plant health, the single-image normalized difference vegetation index (SI-NDVI) compares leaf reflectance in visible and near-infrared light spectrums. METHODS AND RESULTS: The SI-NDVI imaging system was characterized to assess plant responses to stress before visual detection during controlled stress assays. Images were analyzed using Fiji image processing software and Microsoft Excel to create qualitative false color images and quantitative graphs to detect plant stress. CONCLUSIONS: Stress was detected in Arabidopsis thaliana seedlings within 15 min of salinity application using SI-NDVI analysis, before stress was visible. Stress was also observed during ammonium nitrate treatment of Eruca sativa plants before visual detection. Early detection of plant stress is possible using SI-NDVI imaging, which is both simpler to use and more cost efficient than traditional dual-image NDVI or hyper-spectral imaging.

Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight?

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in establishing the spaceflight responses are being identified, their roles in spaceflight physiological adaptation are increasingly understood, and the fact that different genotypes adapt differently is recognized. However, the basic question of whether these spaceflight responses are actually required for survival has yet to be posed, and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses in the root tips of two Arabidopsis ecotypes, Col-0 and WS, as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS, phyD plants displayed a significantly reduced spaceflight transcriptome response compared to Col-0, suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS, more broadly indicating that the plant genotype can be manipulated to reduce the cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could further reduce, or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS, the WS genotype actually mounted a larger transcriptome response than Col-0, suggesting that the in-space light environment affects physiological adaptation, which implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

ARG1 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight.

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight. Key Words: ARG1-Spaceflight-Gene expression-Physiological adaptation-BRIC. Astrobiology 17, 1077-1111.

Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate-binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.