Gravitation Biology
Cellular functions and responses to environmental stimuli are regulated by the activity and interaction of complex molecular networks. Gravity is one of the environmental factors that control development and growth of plants. Studies have shown that the perception of gravity is not only a property of specialized tissues such as the root columella, but can also be performed by undifferentiated cultured cells.
In our experiments we use a homogeneous population of different cell lines of Arabidopsis thaliana for our scientific approaches (transcriptomics, (phospho) proteomics, metabolomics). They are either transgenic or wild-type, callus or suspension cultures.
A comparison of patterns of gene expression (transcriptome) after exposure to hyper-g (centrifugation between 2 and 10g for different periods of time) with 1g controls revealed that hypergravity triggers a general stress response in these cells with a threshold at about 400 gxmin.
Changes in gene expression should – with some delay in time – also result in corresponding alterations of the proteome, which is defined as the protein complement of a particular tissue / cell at a defined condition / state. In accordance with our gene expression studies, where we found significant changes in transcript contents after exposure to 8g for 1 h, we were also able to detect corresponding alterations in the proteome of Arabidopsis cell cultures.
In addition, we compare hypergravity-induced changes with those obtained under simulated weightlessness due to clinorotation (2-D clinostat and 3-D random positioning machine, RPM; magnetic levitation), or real weightlessness as obtained by parabolic flights (20 sec), sounding rocket flights (6 to 12 min), and satellite missions (21 days).
A combination of the separation of single protein spots by 2-D SDS PAGE and subsequent identification by MS (nano HPLC-ESI-MS/MS; in cooperation with the Proteome Center Tübingen) revealed a series of proteins with treatment-specific altered amounts. These in part correspond to the respective transcripts with some delay in time. Functional analysis of identified proteins indicate that changes in the gravitational field induce oxidative stress. Ongoing work focuses on alterations of the phosphoproteom, because fastest responses are to be expected for protein modulation by protein kinases / phosphatases.
Parabolic flight experiments (gravity >10-3g) have shown that the cell cultures respond immediately to changed gravitational forces. Real time in vivo fluorescence measurements demonstratean increase in cytosolic calcium (Ca2+) and hydrogen peroxide (H2O2) with the onset of microgravity.At the same time we measured a decrease in the NADPH/NADP redox ratio. Ca2+ and H2O2 are both important second messenger molecules that trigger subsequent signalling cascades in the cell, resulting in corresponding alterations on the level of gene expression and protein modification.
2D SDS PAGE analysis and liquid chromatography-MS revealed sets of phosphorylated proteins, partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of reactive oxygen species (ROS). Related gene expression analysis (microarray analysis) identified hundreds of differentially expressed transcripts dominated by genes coding for Ca2+- and ROS-related products. During special parabolic flight campaigns with partial-g levels (martian gravity 0.38g; lunar gravity 0.16g and microgravity >10-3g) an exposure to martian gravity was sufficient to induce a gravity response.
During the Simbox/Shenzhou8 mission, a German-Chinese cooperative space project, our experiment was one out of 17 projects that was launched onboard a satellite to a module (Tjangong-1) of the Chinese space station to spend 17 days in space. Whole transcriptome analysis after the mission indicated lesser differences to 1g controls than after shorter times of exposure to micgrogravity. Obviously, the cell cultures adjust to microgravity with time.
The investigation of responses to a lack of gravity opens access to the understanding of gravity-controlled mechanisms on Earth.
Contact & links:
svenja.fengler@uni-tuebingen.de
niklas.hausmann@uni-tuebingen.de
ruediger.hampp@uni-tuebingen.de