A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation

Abstract

Callus cells of Arabidopsis thaliana (cv. Landsberg erecta) were exposed for 8 h to a horizontal clinostat rotation (H, simulated weightlessness), a vertical clinostat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and fructose apparently decreased, while starch content increased in the H compared with the V- and S-treated cells. In order to investigate the influences of clinostat rotation on the cellular proteome further, the proteome alterations induced by horizontal and vertical clinostat rotation have been comparatively analysed by high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 80 protein spots showed quantitative and qualitative variations that were significantly (P <0.01) and reproducibly different between the clinorotated (H or V) and the stationary control samples. Protein spots excised from 2-D gels were analysed by microbe high performance liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. 18 protein spots, which showed significant expression alteration only under the H condition compared with those under V and S conditions, were identified. Of these proteins, seven were involved in stress responses, and four protein spots were identified as key enzymes in carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated in the H samples. Other proteins such as protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins also exhibited altered expression under the H condition. The data presented in this study illustrate that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins involved in general stress responses, metabolic pathways, gene activation/transcription, protein synthesis, and cell wall biosynthesis.

Gene expression changes in Arabidopsis seedlings during short- to long-term exposure to 3-D clinorotation

“…Kittang et al (2004) performed DNA chip experiments for profiling of the genes with altered expressions under simulated microgravity condition (1 or 24 h) and found out that about 2.2% of total 8,000 genes screened showed differential expression levels. The experiment was done for cultured Arabidopsis cells to monitor the contents of translational gene products in response to simulated microgravity condition (clinorotation) (Wang et al 2006;Barjaktarovic et al 2007). In this case, proteome of the exposed undifferentiated plant cells (calli) mounted a significant response to simulated microgravity conditions within a period of 10 min to 16 h. Furthermore, these earlier studies provide no information about time-course based response pattern of the plant genes from short-to long-term exposure to simulated microgravity.…”

Alterations in Protein Expression of Arabidopsis thaliana Cell Cultures During Hyper- and Simulated Micro-Gravity

“…This tendency starts already at 4 h of exposure. We assume, that these changes are more related to metabolic adaptation, as previously reported for A. thaliana callus cells exposed to 8 h of horizontal 2-D clinorotation (Wang et al 2006). In the latter study, 18 proteins were detected to be altered in expression, some of them being involved in carbon and lipid metabolism.…”

“…such changes should-with some delay in time-also affect protein expression patterns. Accordingly, a recent study showed that clinorotation alters protein patterns as obtained by 2-D-SDS-PAGE of soluble proteins (Wang et al 2006). Identification of some spots indicated that these proteins were involved in carbohydrate and lipid metabolism, signalling, gene expression, and cell wall biosynthesis.…”

Clinorotation Affects the Ultrastructure of Pea Root Mitochondria

“…There is a number of data on the changes of cell functioning in microgravity and under clinorotation (Halstead and Dutcher 1987;Kordyum 1997;Baranenko 1998;Kozeko and Kordyum 2006;Wang et al 2006), and it is assumed that actively metabolizing cells are the most sensitive to altered gravity (Kordyum 1997). A root DEZ is characterized by specific physiological properties.…”