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RESEARCH PAPER |
Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures
arko Barjaktarovi
1
1University of Tübingen, Botany Institute, Physiological Ecology of Plants, Auf der Morgenstelle 1, D-72076 Tübingen, Germany
2University of Tübingen, Interfaculty Institute for Cell Biology, Proteom Centrum Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany
* To whom correspondence should be addressed. E-mail: ruediger.hampp{at}uni-tuebingen.de
Received 14 September 2007; Revised 2 November 2007 Accepted 5 November 2007
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Abstract |
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In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS–PAGE. Image analysis of Sypro Ruby®-stained gels showed that
28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning. Key words: Arabidopsis thaliana, cell cultures, hypergravity, proteomics, simulated microgravity
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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 (Chen et al., 1999; Rosen et al., 1999). 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 (Kiss et al., 1989; Sack, 1997; Blancaflor et al., 1998; Martzivanou and Hampp, 2003; Babbick et al., 2006). A comparison of patterns of gene expression (transcriptome) of Arabidopsis cell cultures after exposure to hyper-g (centrifugation between 2 g and 10 g for different periods of time) with 1 g controls revealed that hypergravity triggers a general stress response in these cells, with a threshold at
400 gxmin (Martzivanou and Hampp, 2003). Out of the up-regulated transcripts of 200 genes, 12% were involved in signalling and phosphorylation/dephosphorylation, 6% in defence and stress response, and 2% in gravisensing. A large fraction of the up-regulated transcripts (17%) coded for gene products involved in cellular organization and cell wall formation/rearrangement. Comparable studies with intact Arabidopsis plants showed that changes in the gravitational vector (tilting of seedlings; clinorotation; centrifugation: 5 g) can cause rapid changes in gene expression (Moseyko et al., 2002; Centis-Aubay et al., 2003; Kimbrough et al., 2004). In the study by Kimbrough et al. (2004), transient changes in the relative abundance of genes were found within 2 min of stimulation. This included genes involved in the regulation of sterol-, brassinosteroid-, and jasmonate-related signal transduction pathways. 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 previous gene expression studies, where significant changes in transcript contents after exposure to 8 g for 1 h were found, time- course experiments were performed between 30 min and 16 h at 8 g in order to monitor alterations in the proteome of Arabidopsis cell cultures. In addition, data are reported which compare hypergravity-induced changes with those obtained under simulated weightlessness due to clinorotation (2-D clinostat and 3-D random positioning machine, RPM). A combination of the separation of single protein spots by 2-D SDS–PAGE and subsequent identification by mass spectrometry [nano high-pressure liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS)] revealed 28 proteins with treatment-specific altered amounts. These in part corresponded to the respective transcripts with some delay in time. Functional analysis of identified proteins indicates that changes in the gravitational field induce oxidative stress.
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Materials and methods |
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Cell cultures
Cell suspension cultures were generated from leaves of Arabidopsis thaliana (cv. Columbia) plants, grown under sterile conditions. Calli obtained on 1% agar plates containing I.2a medium (Seitz and Alfermann, 1985) were transferred to a liquid I.2a medium (20 ml without agar in 500 ml Erlenmeyer flasks) and grown at 26 °C in the dark on a rotary shaker (Infors, Bottmingen, Switzerland; 13 rpm). New medium was added every week. This suspension culture was taken as stock for repeated callus formation. For this purpose, cell suspensions (10 ml) were spread on 9 cm plates containing agar/I.2a medium as above, with the surplus of medium being decanted. Calli with a diameter of
1 mm were obtained after 1 week of growth and used for the experiments. At the end of exposure, calli were rapidly scraped off the agar (<5 s), submerged in liquid nitrogen, and stored at –80 °C until use. For each time point, corresponding control samples were collected, which were exposed in close vicinity to the exposed samples (inside the centrifuge housing; in the temperature-controlled RPM room; same table as the 2-D clinostat).
Application of hypergravity (8 g), clinorotation, and random positioning
Application of 8 g was by centrifugation of the Petri dishes. The radius (distance between the centre of the centrifuge axis and the centre of the Petri dish) was determined to adjust the corresponding rpm number. Petri dishes were fixed in holders the angle of which could be adjusted by micrometer screws. The angle was set such that the resulting centrifugal force was applied perpendicular to the Petri dish surface.
Clinorotation was performed at 60 rpm, with tubes having an internal diameter of 1 cm. This arrangement resulted in a maximal gravitational force of 0.0016 g (DLR Hemmersbach, personal communication). The 12 ml screw-capped tubes (Greiner BioOne, Nürtingen, Germany) were positioned horizontally and filled with 6 ml of 1% agar. In this way, the agar surface corresponded to the inner diameter of the tube. On this surface, 100 mg of 7-d-old cultures were spread.
For random positioning, cultures were prepared on Petri dishes as described above for centrifugation. The dishes were then fixed in the centre of the inner of the two connected frames. The frames were rotating in a random, autonomous way at an angular velocity of 60 ° s–1 (Walther et al., 1998). For details of the different procedures, see also Babbick et al. (2006).
Protein extraction
The extraction procedure is based on the method of Ninii et al. (1996) with some modifications. A 1 g aliquot of fresh weight of Arabidopsis calli was ground to a fine powder in liquid nitrogen and transferred to 2 ml of lysis buffer [40 mM TRIS-HCl, pH 8, containing both plant protease inhibitor cocktail and phosphatase inhibitor cocktail II according to the manufacturer's suggestions (20 µl each; Sigma)]. The suspensions were kept on ice for 30 min with gentle vortexing every 10 min. Then, 2.5 ml of water-saturated phenol were added, and the samples were subjected to a freeze (liquid nitrogen)–thaw cycle, and shaken at 4 °C for 1 h. After centrifugation at 10 000 rpm for 10 min, the phenol phase was washed twice with 2 ml of lysis buffer (see above). Proteins were precipitated with 3 vols of 10% trichloroacetic acid in acetone for 1 h at –20 °C and centrifuged at 15 000 rpm for 2 h. The pellet was washed twice with 1 ml of acetone, and resolubilized in a buffer containing 9.5 M urea (increased solubilization), 60 mM dithiothreitol (DTT), 2% CHAPS, and 0.5% ampholines, pH 3–10 (GE Healthcare, Munich, Germany). Protein concentration was determined using the Bradford assay (Bio-Rad, Munich, Germany) with bovine serum albumin (BSA) as standard.
2-D gel electrophoresis and image analysis
Proteins were separated by 2-D SDS–PAGE using immobilized pH gradients [18 cm pH gradient polyacrylamide gel strips in the pH range of 4–7 (Bio-Rad)]. An 80 µg aliquot of total proteins in rehydration buffer (8 M urea, 2% CHAPS, 30 mM DTT, 0.5% ampholines pH 4–7) was loaded onto the gel strips in a Protean IEF Cell (Bio-Rad). Isoelectric focusing was carried out at 20 °C, using voltages and running times as follows: 12 h passive rehydration, 300 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3500 V for 2 h, 8000 V for 2 h, 10 000 V for 3–4 h (for a total 55 kVh). Maximum current was 50 µA per gel strip. Gel strips were incubated in equilibration solution [50 mM TRIS-HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 1% (w/v) DTT] for 15 min, followed by a second 15 min incubation with the same solution, but DTT substituted by 2.5% (w/v) iodoacetamide. Equilibrated gel strips were placed on top of a 12% acrylamide gel (PDA as a cross-linker, Bio-Rad) and overlaid with 0.5% agarose solution. SDS–PAGE was carried out using the large Protean II Cell (Bio-Rad), and was performed at 20 mA for 1 h, followed by 40 mA for 5 h. Gels were then placed in 250 ml of fixing solution (7% acetic acid, 10% methanol), and stained in 250 ml of Sypro Ruby solution (Bio-Rad) overnight. After washing twice for 1 h in fixing solution, the gels were recorded using the Gel Doc 2000 system (Bio-Rad). Differences in protein expression were analysed using PD QUEST, version 8.1 (Bio-Rad). The significance of expression changes was determined by Student's t-test at a significance level of 95% (
1.5-fold increased or 
0.66-fold decreased).
Each protein preparation was subjected to 2-D PAGE at least in triplicate, and three independent protein preparations were made for each type of treatment or time of exposure.
Spots were excised manually from the gel and digested with trypsin (sequencing grade, Promega, Mannheim, Germany). Eluted peptides were analysed using a Dionex LC Packings nano-HPLC system (Idstein, Germany), containing the components FAMOS (autosampler), Switchos (loading pump and switching valves), and Ultimate (separation pump and UV detector). The ESI-MS/MS spectra were recorded as detailed elsewhere (Resch et al., 2006).
Analysis of spectrometric data
Proteins were identified by correlating the ESI-MS/MS spectra with the NCBI.nr-protein sequence database of A. thaliana as of 8 June 2007 using the MOWSE-algorithm as implemented in the MS search engine MASCOT (Matrix Science Ltd, London, UK; Perkins et al., 1999). All experimental data, achieved by 2-D gel electrophoresis and mass spectrometry, and corresponding search results were stored in a LIMS database (Proteinscape 1.3, Bruker Daltonics, Bremen, Germany).
Database search results were reviewed according to the following criteria: (i) number of matched peptides (must be 2); (ii) MASCOT score [probability-based MOWSE score: –10xLog(P), where P is the probability that the observed match is a random event. Scores >34 indicate identity or extensive homology; P <0.05]; and (iii) sequence coverage (10%). Additionally, every peptide used for protein identification was checked for (i) y-ion series (80% of the y-ions should be available); (ii) presence of the b2-ion; (iii) peptide score (>15); and (iv) e-value (probability that the observed match is a random peptide). In the case of more than one protein meeting the criteria, only the protein with the most significant MASCOT score is shown. Identified proteins were functionally annotated via the MIPS database.
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Protein isolation and 2-D gel separation
After 2-D SDS–PAGE, gels were stained by Sypro Ruby for spot visualization and comparison between treatments. About 800 spots could be reproducibly identified with PD Quest software (Fig. 1). After 2 h of centrifugation, 28 spots were changed in amount by >1.5-fold, or <0.66-fold with respect to control. These spots were excised from Colloidal Coomassie Blue-stained gels and analysed by ESI-MS/MS.
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Responses to hypergravity (8 g)
After 2 h of exposure, 18 out of the 28 identified proteins were increased in abundance (Table 1). These are involved in different stress responses/detoxification reactions (HSP91, methionine synthase, methionine sulphoxide reductase, aldehyde dehydrogenase, FQR1, aconitase, CR88), protein folding (chaperones; HSP60, HSP91, calnexin, annexin, protein disulphide isomerase), regulation of gene expression (MBD10), cell division plate formation (CDC48), photomorphogenesis (AJH1, CR88), and metabolism [CPN60, alanine aminotransferase, lipoamide dehydrogenase, aconitase, aldolase, malate dehydrogenase (NADP)].
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Out of these, CDC48, CPN60, FQR1, and aldolase (FBP) decreased after 2 h and reached their minimum amount between 4 h and 8 h of treatment. After 16 h of exposure, they were elevated again (data not shown). This implies that certain steps of cytokinesis, enzyme assembly, auxin responses, and glycolysis/gluconeogenesis are transiently down-regulated after an initial rise.
Proteins such as MBD10, HSP91, ATP-binding protein, an unknown protein, and, to some extent, also methionine sulphoxide reductase behaved in an opposite way. They continued to increase and reached their maximum amount between 4 h and 8 h (data not shown). These changes could be indicative of an increased transcriptional repression (MBD10) and partially increased stress (heat shock, oxidative) responses.
A third group which is increased in amount after 2 h (aldehyde dehydrogenase, protein disulphide isomerase, calnexin, and NADP malate dehydrogenase) exhibited a steady decline under prolonged exposure. These proteins are involved in detoxification reactions (oxidative stress) and primary metabolism.
Finally, there is a group of proteins which were down-regulated after 2 h of exposure (between 39% and 75% of 1 g controls; glutathione S-transferase, lipoamide dehydrogenase, methionine synthase, aconitase, aminopeptidase, HSP60, and ATP synthase; Table 1). Most of the proteins which were altered in amount after 2 h returned to control levels after 16 h of exposure. Only lipoamide dehydrogenase and ATP synthase exceeded controls after 16 h (data not shown). Physiological functions of these proteins can be associated with detoxification, primary/amino acid/ethylene metabolism, and protein folding/mitochondrial biogenesis.
Responses upon exposure to 2-D clinorotation
To a considerable extent (19 out of 28 proteins), alterations in protein abundance after 2-D clinorotation are comparable with those under hypergravity (8 g/2D; ratios after 2 h of treatment >1.5 or <0.5; Table 1). Differences are most obvious for HSP60 (assembly of multimeric proteins, activation of mitochondrial biogenesis) which is up-regulated upon clinorotation but down-regulated under centrifugation; the opposite was found for inorganic diphosphatase (energy supply for, for example, vacuolar ATPases) and ATP-binding protein.
Responses induced by random positioning
RPM exposure caused considerably fewer changes in amounts of proteins. Only eight out of the 28 proteins affected by hyper-g and 2-D were altered (Table 1). Generally, and with the exception of HSP91, responses were weaker in comparison with the other treatments. Some similarities in behaviour existed for aminopeptidase, AJH1, MBD10 (all 8 g, 2D), HSP60, alanine aminotransferase (both 2-D), HSP91, and protein disulphide isomerase (both 8 g).
Only aldehyde dehydrogenase (detoxification; down-regulated) and aconitase [tricarboxylic acid (TCA) cycle; increase after initial down-regulation] showed changes in abundance which were different from those of either 8 g or 2D clinorotation (Table 1b).
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Discussion |
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Some of the altered proteins (methionine sulphoxide reductase, annexin, aldehyde dehydrogenase, FQR1, as well as proteins with chaperone function) are involved in detoxification, especially of reactive oxygen species (ROS). Oxidative stress is a component of most abiotic stresses (Apel and Hirt, 2004), and plant cells can control ROS production for the purpose of signalling. This is achieved by establishing a balance between production and scavenging of ROS (Pitzschke et al., 2006). A surplus of ROS will cause oxidative damage to cellular functions. Plants thus possess a large number of antioxidant enzymes and metabolites (Mittler et al., 2004). If the capacity of the cellular antioxidative system cannot cope with the production of ROS, this eventually leads to cell death (Halliwell, 2006). ROS are, however, also a component of stress signalling, which can be modulated by plant cells (Halliwell, 2006).
With this background, the present data could thus indicate that A. thaliana cell cultures perceive changes in the gravitational field strength as abiotic stress. Owing to the transient nature of the responses, it is assumed that gravitation-related signalling uses ROS. Such an assumption is supported by findings that show that ROS are involved in gravitropic root curvature (Joo et al., 2001)
In an earlier study (array with 4100 genes; Martzivanou and Hampp, 2003), where changes in gene expression under hypergravity were studied, 200 were found to be significantly elevated in amount after 60 min. From these, nine can be related to the proteins identified in this study after 2 h of exposure to hypergravity [CDC48, aldehyde dehydrogenase, malate dehydrogenase, fructose bisphosphate aldolase, calnexin, glutathione transferase, aconitate hydratase ( = aconitase), methionine synthase, and HSP60]. These are involved in detoxification and metabolism. For the other transcripts found to be altered in amount in Martzivanou and Hampp (2003), the respective proteins could not be detected. This could have several reasons such as lack of translation, turnover of transcripts/proteins, amounts too low to be detected, or the array used. The latter assumption seems most reasonable because an early array (IncyteGenomics) was used with only 4105 unique annotated clusters/genes, compared with >25 000 available recently.
In addition to enzymes that are involved in detoxification, others such as aconitase are known to be sensitive to oxidative inhibition. Upon treatment with H2O2 and menadione, Sweetlove et al. (2002) reported decreased spot intensities for mitochondrial proteins, including ATP synthase, complex I, succinyl CoA ligase, aconitase, and others. Obviously, oxidative inhibition of these proteins is connected to protein degradation. Thus, the decrease in protein amounts found for ATP synthase and aconitase is another indication of a stress response including ROS. Baxter et al. (2007) reported that cell cultures of A. thaliana exhibited metabolic responses, which indicated that enzymes of the TCA cycle and of glycolysis were repressed, while those belonging to the oxidative pentose phosphate pathway (OPPP) were induced (redirection of carbon from respiration to the OPPP for NADPH production). In addition, enzymes of the polyamine pathway and those involved in the synthesis of a range of amino acids were repressed under oxidative conditions.
There is only little indication for such a shift. In the samples used in the present experiments, this is mainly confined to the amounts of aconitase (TCA) and the decrease in the amount of methionine synthase (methionine is a precursor for the synthesis of polyamines). Probably the effects induced in the experiments are considerably weaker than those reported for the menadione treatment.
Interestingly, the responses discussed so far are primarily related to centrifugation and 2-D clinorotation, while samples exposed to random positioning show weaker effects. The unexpected similarities between hypergravity and clinorotation could be due either to residual gravity or to permanent stimulation due to rotation. According to calculations by Dr Ruth Hemmersbach (ruth.hemmersbach{at}dlr.de), the cell cultures experienced only 0.0016 g at most (60 rpm; 4 mm radius) which is below the gravity response threshold (e.g. 0.16 g for Euglena cells; Häder and Hemmersbach, 1997; 0.05 g for the displacement of statoliths in Chara rhizoids; Limbach et al., 2005). Thus it is at least similarly likely that permanent stimulation is the cause for similarities in the stress response. In contrast, the RPM-specific responses (aldehyde dehydrogenase, detoxification, down-regulated; and aconitase, TCA cycle, increase after initial down-regulation) could be interpreted as a decrease in ROS stress in comparison with 1 g samples [there is also obviously always a basic ROS load in unstressed cells which could regulate the redox state (Halliwell, 2006)]. Probably, changes in position/orientation are so fast that the cells ‘forget’ about the gravity vector.
Random positioning is increasingly used for microgravity simulation (Kittang et al., 2004; Infanger et al., 2006; Meloni et al., 2006). As far as comparable data exist, RPM treatments show similarities to real microgravity as experienced in sounding rocket experiments (M Babbick, M Cogoli, and R Hampp; unpublished data) or space shuttle experiments (Marco et al., 2006).
When the means are calculated for all treatments where the values for the 3D/control ratios also show a response (ratios other than 1.0; last three columns of Table 1b), then five proteins show a change 1.5-fold (values in parentheses). These are AJH1 (1.9), alanine aminotransferase (1.5), MBD10 (1.5), malate dehydrogenase (1.6), annexin (2.3), and HSP91 (2.3). In addition, the putative aminopeptidase is down-regulated by 0.5-fold. These proteins respond to environmental stimuli and indicate that their expression is obviously sensitive to all of the treatments applied.
The only other study on gravitational field-induced changes of the proteome was published by Wang et al. (2006). These authors also used A. thaliana callus cells (in this case the cv. Landsberg erecta) which were exposed for 8 h to horizontal and vertical clinostat rotation (10 rpm, 2.5 cm diameter; 2.8x10–3 g), in comparison with stationary growth. Horizontal clinorotation resulted in significant changes in the amount of 18 proteins. Most of them were up-regulated. Of these, seven are involved in stress responses, and four can be related to carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated. Other proteins altered in amount were protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins. When these data are compared with the 8 h/2-D clinorotation data, only homologies for aldehyde dehydrogenase and glutathione S-transferase were found, which both are increased in amount. A possible reason for this difference could be the experiment design. Proteins which were altered in amount after 2 h of exposure were screened for, and changes of these proteins were followed over time. Wang et al. (2006), in contrast, recorded differences in the proteome after 8 h of exposure. Under the assumption that both A. thaliana systems/exposures can be compared, then the present data reflect the protein complement shortly after changing steady-state conditions, while those of Wang et al. (2006) reflect more those after reaching a new steady state. Consequently, in the present data, stress responses dominate, while the protein changes reported by Wang et al. (2006) could be more related to metabolic adaptation. This assumption is supported by gels where 2-D separations after 2 h and 8 h of exposure to 8 g are compared (data not shown). After 8 h there is clearly a different set of proteins altered in amount. These spots have not, however, been identified yet.
In conclusion, it is assumed that earth's gravitation could impose some basic stress response which is relieved by reducing the gravitational field. This is a working hypothesis for further experimentation.
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Acknowledgements |
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We gratefully acknowledge helpful suggestions by Dr Elisabeth Magel (University of Hamburg), Dr Mika Tarkka (Helmholtz-Centre for Environmental Research, Halle), and Dr Ruth Hemmersbach (DLR, Köln). The investigation was made possible by a grant from the Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR; grant no. 50WB0423)
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