PREMISE OF THE STUDY: Gravity has been a major force throughout the evolution of terrestrial organisms, and plants have developed exquisitely sensitive, regulated tropisms and growth patterns that are based on the gravity vector. The nullified gravity during spaceflight allows direct assessment of gravity roles. The microgravity environments provided by the Space Shuttle and International Space Station have made it possible to seek novel insights into gravity perception at the organismal, tissue, and cellular levels. Cell cultures of Arabidopsis thaliana perceive and respond to spaceflight, even though they lack the specialized cell structures normally associated with gravity perception in intact plants; in particular, genes for a specific subset of heat shock proteins (HSPs) and factors (HSFs) are induced. Here we ask if similar changes in HSP gene expression occur during nonspaceflight changes in gravity stimulation. METHODS: Quantitative RT-qPCR was used to evaluate mRNA levels for Hsp17.6A and Hsp101 in cell cultures exposed to four conditions: spaceflight (mission STS-131), hypergravity (centrifugation at 3 g or 16 g), sustained two-dimensional clinorotation, and transient milligravity achieved on parabolic flights. KEY RESULTS: We showed that HSP genes were induced in cells only in response to sustained clinorotation. Transient microgravity intervals in parabolic flight and various hypergravity conditions failed to induce HSP genes. CONCLUSIONS: We conclude that nondifferentiated cells do indeed sense their gravity environment and HSP genes are induced only in response to prolonged microgravity or simulated microgravity conditions. We hypothesize that HSP induction upon microgravity indicates a role for HSP-related proteins in maintaining cytoskeletal architecture and cell shape signaling.
Research Containing: Genes
The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.
Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq
A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.
The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.
MIZ1, an essential protein for root hydrotropism, is associated with the cytoplasmic face of the endoplasmic reticulum membrane in Arabidopsis root cells
MIZ1 is encoded by a gene essential for root hydrotropism in Arabidopsis. To characterize the property of MIZ1, we used transgenic plants expressing GFP-tagged MIZ1 (MIZ1-GFP) and mutant MIZ1 (MIZ1(G235E)-GFP) in a miz1-1 mutant. Although both chimeric genes were transcribed, the translational products of MIZ1(G235E)-GFP did not accumulate in roots. Moreover, MIZ1-GFP complemented the mutant phenotype but not MIZ1(G235E)-GFP. The signal corresponding to MIZ1-GFP was detected at high levels in cortical cells and lateral root cap cells and accumulated in compartments in cortical cells. MIZ1-GFP was fractionated into a soluble protein fraction and an endoplasmic reticulum (ER) membrane fraction, where it was bound to the surface of the ER membrane at the cytosolic side.
Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots
The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.
Epstein-Barr virus (EBV) latent and replicative gene transcription was analyzed in peripheral blood B-lymphocytes from astronauts who flew on short-duration ( approximately 11 days) Shuttle missions and long-duration ( approximately 180 days) International Space Station (ISS) missions. Latent, immediate-early, and early gene replicative viral transcripts were detected in samples from six astronauts who flew on short-duration Shuttle missions, whereas viral gene transcription was mostly absent in samples from 24 healthy donors. Samples from six astronauts who flew on long-duration ISS missions were characterized by expanded expression of latent, immediate-early, and early gene transcripts and new onset expression of late replicative transcription upon return to Earth. These data indicate that EBV-infected cells are no longer expressing the restricted set of viral genes that characterize latency but are expressing latent and lytic gene transcripts. These data also suggest the possibility of EBV-related complications in future long-duration missions, in particular interplanetary travel.
Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments
Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 muM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 muM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.
Overexpression of MIZU-KUSSEI1 enhances the root hydrotropic response by retaining cell viability under hydrostimulated conditions in Arabidopsis thaliana
Because of their sessile nature, plants evolved several mechanisms to tolerate or avoid conditions where water is scarce. The molecular mechanisms contributing to drought tolerance have been studied extensively, whereas the molecular mechanism underlying drought avoidance is less understood despite its importance. Several lines of evidence showed that the roots sense the moisture gradient and grow toward the wet area: so-called hydrotropism. We previously identified MIZU-KUSSEI (MIZ) 1 and MIZ2/GNOM as genes responsible for this process. To gain new insight into the molecular mechanism of root hydrotropism, we generated overexpressors of MIZ1 (MIZ1OEs) and analyzed their hydrotropic response. MIZ1OEs had a remarkable enhancement of root hydrotropism. Furthermore, a greater number of MIZ1OE root cells remained viable under hydrostimulated conditions than those of the wild type, which might contribute to retaining root growth under hydrostimulated conditions. Although overexpression of MIZ1 also caused a slight decrease in the root gravitropic response, it was not attributable to the enhanced hydrotropic response. In addition, miz2 mutation or the auxin response inhibitor nullified the enhanced hydrotropic response in MIZ1OEs. Furthermore, the expression of MIZ1 did not alter the expression of typical genes involved in drought tolerance. These results suggest that MIZ1 positively regulates hydrotropism at an early stage and its overexpression results in an enhancement of signal transduction unique to root hydrotropism to increase the degree of hydrotropic root bending.
Mechanical load induces upregulation of transcripts for a set of genes implicated in secondary wall formation in the supporting tissue of Arabidopsis thaliana
We examined the effects of mechanical load on transcripts of a set of cell wall related genes that are implicated in the formation of supporting tissues, by applying a 50 mg strip of aluminum foil to the inflorescence stem of Arabidopsis thaliana, a weight roughly half the fresh weight of the stem. Transcript levels of 12 of the 15 genes examined were increased by load application, as were the levels of some transcription factors that regulate secondary wall formation. These findings support the involvement of a load-sensing system in regulation of supporting tissue formation via transcriptional regulation of cell wall related genes.