JXB Advance Access originally published online on January 31, 2006
Journal of Experimental Botany 2006 57(4):827-835; doi:10.1093/jxb/erj066
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RESEARCH PAPER |
A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation
1Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
2Research Center for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200032, China
* To whom correspondence should be addressed. E-mail: hqzheng{at}sippe.ac.cn
Received 2 August 2005; Accepted 18 November 2005
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionConcluding remarksSupplementary dataReferences |
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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.
Key words: Arabidopsis thaliana, callus cells, clinostat rotation, proteomics
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksSupplementary dataReferences |
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Gravity has a profound influence on plant growth and development. Removal of the influence of gravitational acceleration by spaceflight causes a wide range of cellular changes in plants (Cogoli and Gmünder, 1991
; Hampp et al., 1997). Since the opportunity to study the role of gravity in space is limited, clinostats have been developed to partially simulate, in ground-based experiments, the weightless environment of space. Growing plants on the clinostat with a horizontal axis corresponds to constant omnilateral gravity stimulation (Brown et al., 1976; Smith et al., 1997). Whole seedlings, germinated and grown on clinostats, show an absence of certain features that are characteristic of gravitropically-grown plants (Shen-Miller and Gordon, 1967; Stankovi
et al., 2001). At the cellular level, clinostat treatments produce specific effects on plant cells, such as alterations in cell wall composition, increased production of heat-soluble proteins, and changes in cellular energy metabolism (reviewed by Claassen and Spooner, 1994; Shen-Miller and Hinchman, 1995; Vasilenko and Popova, 1996). Other changes include a more random distribution of plastids (amyloplasts) and an increase in the number and volume of nucleoli (Shen-Miller and Hinchman, 1995; Smith et al., 1997). Protoplasts of Brassica napus exposed to both microgravity and clinostat rotation exhibited a decrease in the cell divisions and abnormal microtubule arrays (Skagen and Iversen, 2000).
A number of recent studies have shown that the exposure of Arabidopsis seedlings and callus cells to gravity stimulation, hyper g-forces or clinostat rotation induces alterations in gene expression (Moseyko et al., 2002; Centis-Aubay et al., 2003; Martzivanou and Hampp, 2003; Yoshioka et al., 2003; Kimbrough et al., 2004). These studies confirmed the findings of several earlier studies that showed that altered gravity conditions led to the synthesis of new proteins in the root tips of corn and Arabidopsis seedlings (Feldman, 1983; Feldman and Gildow, 1984; Friedmann and Poovaiah, 1991). However, the findings of Rasmussen et al. (1992), who reported a major decrease of total protein content and the loss of distinct bands from the electrophoresis pattern in rapeseed callus grown in orbit, should not be overlooked.
Proteome analysis provides a means for analysing differential gene expression at the protein levels, and this approach has proved to be a powerful tool for analysing the responses of plants to environmental stresses, including drought, salt, and high and low temperatures (de Vienne et al., 1999; Salekdeh et al., 2002; Bae et al., 2003; Majoul et al., 2004; Yan et al., 2005). The current study extends this type of analysis to plants subjected to altered gravity conditions. To this end, the proteomes of Arabidopsis callus cells exposed to horizontal clinostat rotation (H), vertical clinostat rotation (V) and stationary control (S) growth conditions have been characterized. The functional implications of the observed changes in protein expression in response to clinostat rotation are discussed.
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Materials and methods |
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Plant materials and cell culture conditions
Callus cultures were produced from Arabidopsis thaliana (cv. Landsberg erecta) seeds in MS medium (Murashige and Skoog, 1962
) supplemented with 3% sugar and 2 mg l–1 2,4-D. The suspension cultures were grown in 250 ml conical flasks in the dark at 22–25 °C in an orbital shaker (130 rpm) and were subcultured every week. The suspension cultures were taken as stock for repeated callus formation. At 6–7 d after subculture, the cultures were transferred to solid medium supplemented with 1% agar (as described by Martzivanou and Hampp, 2003). Calli with a diameter of about 1 mm were used for experiments after 1 week of growth on Petri dishes.
Clinostat treatments
A one-axis 1-
clinostat facility (SB-01) was used to simulate weightlessness. Characteristic physical details of this instrument were given by Liu et al. (1993). Briefly, this device was designed as two groups of orthogonal axes allowing simultaneous rotation around each of four horizontal and vertical axes. The rotation rate of the clinostat (0.5–200 rpm) could be adjusted and controlled by a control box. The clinostat rotation treatment in this study consisted of a clinostat oriented horizontally (H) and rotating at 10 rpm (e.g. the level of acceleration applied to the materials was less than 2.8x10–3 g at a radius of 2.5 cm). In addition to the stationary 1 g control (S), a vertically oriented clinostat (V) rotating at 10 rpm was used as a control to evaluate potential mechano-stimulation artefacts from the clinostat motor and the side-effects of rotation itself. For each experiment, three calli (fresh weight 80–100 mg each) were grown at the same distance (2.5 cm) to a central dot on Petri dishes, and the Petri dishes were mounted on the clinostat as described by Liu et al. (1993).
Carbohydrate analysis
About 10 mg of the callus cells were incubated in 80% ethanol at 60 °C for 1 h, dried, and extracted in 200 µl ddH2O at room temperature for 15 min with occasional vortexes, followed by centrifugation at 10 000 g for 10 min. The supernatants were used for soluble sugar assays according to Hampp et al. (1994). The absorbance was recorded on a microplate reader system (Sunrise Tecan, USA). The starch of the pellets was determined as previously described by Obenland and Brown (1994). Five independent samples were measured to obtain the mean values ±SE.
Protein extraction and 2-DE analysis
1 g of calli were ground in liquid nitrogen to a fine powder and resuspended in an ice-cold solution of 10% w/v trichloroacetic acid (TCA) in acetone with 0.07% w/v DTT for at least 1 h at –20 °C, and centrifuged for 30 min at 35 000 g. The pellets were rinsed twice with acetone containing 0.07% w/v DTT for 1 h at –20 °C and then lyophilized. The resulting powder pellet was solubilized in lysis buffer (7 mol l–1 urea, 2 mol l–1 thiourea, 4% CA-630, 32 mmol l–1 TRIS-HCl pH 6.8, 1 mmol l–1 PMSF, 14 mmol l–1 DTT, and 0.2% Triton X-100) for 1 h at 17 °C and then centrifuged at 12 000 g for 15 min. The proteins in the supernatant were precipitated by adding four volumes of ice-cold acetone, incubated at –20 °C for at least 2 h, and centrifuged at 12 000 g for 15 min. The pellets were dissolved in rehydration buffer containing 8 mol l–1 urea, 2% w/v CHAPS, 18 mmol l–1 DTT, 0.5% w/v IPG buffer pH 4–7 and a trace of bromophenol blue. The protein concentrations were quantified using the Bradford method (Bradford, 1976). Three samples per treatment (H, V and S) were prepared from individual cell cultures. All samples were stored at –80 °C prior to electrophoresis.
2-DE was performed primarily according to Yu et al. (2000). One-dimensional isoelectric focusing (IEF) was performed on an immobilized pH gradient strip (IPG strip; pH4-7, linear, 13 cm, Pharmacia). 100 µg and 800 µg of total proteins were loaded onto analytical and preparative gels, respectively. IEF was conducted in three steps: 500 V for 1 h, 1000 V for 1 h, and 8000 V for 6 h. Focused strips were equilibrated twice for 15 min in 2 ml equilibration solution. The first equilibration step was performed in a solution containing 6 mol l–1 urea, 30% glycerol, 2% SDS, 2% DTT, 50 mmol l–1 TRIS-HCl buffer (pH 8.8). The second equilibration step was performed in a solution modified by the replacement of DTT by 2.5% iodoacetamide. Two-dimensional SDS-PAGE (2-DE) was performed with 1 mm thick, 12.5% T SDS- polyacrylamide gel (Hofer SE 600 in a vertical slab). The gels were run at 10 mA per gel for the first 30 min and followed by 25 mA per gel. After 2-DE, the analytical gels were stained with ammoniacal silver nitrate according to the procedure described by Yu et al. (2001), and the preparative gels were stained with colloidal Coomassie Brilliant blue G-250 (Bradford, 1976). At least three replicates were performed for each sample.
Image acquisition and analysis
The silver-stained 2-D gels were scanned using a GS710 imaging densitometer (Bio-Rad) in transmissive mode. Spot detection, quantification, and matching were performed using PDQuest 7.1 software (Bio-Rad). A matchset consisting of 15 images, five for the H samples, five for the V samples, and five for the S samples was created, and one image from S was selected as the matchset standard for spot matching. The match rate of every gel to the selected standard gel was higher than 90% according to the analytical results by PDquest 7.1 software. Only those with significant and reproducible changes were considered to be differentially accumulated proteins. The abundance of each protein spot was estimated by the percentage volume, for example, the individual spot volumes were normalized by dividing their optical density (OD) values by the total OD values of all the spots present in the gel, and expressed as % Vol. The significance of expression differences of protein spots between clinostat rotational conditions (H or V) and stationary control (S) was estimated by Student's t-test, P <0.01.
In-gel digestion
Protein spots were excised from the preparative gels, rinsed with water (Milli-Q) three times, destained twice with 25 mmol l–1 NH4HCO3 in 50% acetonitrile (ACN), reduced with 100 mmol l–1 DTT in 100 mmol l–1 NH4HCO3, alkylated with 200 mmol l–1 iodoacetamide in 100 mmol l–1 NH4HCO3, dried by lyophilization, and digested for 20 h at 37 °C with 5 ng µl–1 trypsin in 25 mmol l–1 NH4HCO3, pH 8.3. The peptides were extracted three times with 0.1% trifluoroacetic acid (TFA) in 60% ACN. The supernatants were pooled and lyophilized for mass spectrometric analysis.
LC-IT-MS and protein identification
In-gel digested samples were re-dissolved in 0.1% v/v TFA and desalted using a C18 Zip Tip (Millipore). The separation and identification of the peptide mixtures was conducted by a Finnigan LCQ Deca XP ion-trap micro-electrospray mass spectrometer (ThermoQuest, San Jose, CA, USA) coupled with a reversed-phase high-performance liquid chromatography (HPLC) system (ThermoQuest). For LC separation, a 0.15x120 mm column (RP-C18, ThermoHypersil, San Jose, CA, USA) was used. Solvent A was 0.1% v/v formic acid and solvent B was 0.1% v/v formic acid in 100% v/v ACN. The gradient was held at 2% v/v solvent B for 15 min, and increased linearly to 98% v/v solvent B in 90 min. The peptides were eluted from a C18 microcapillary column at a flow rate of 150 µl per min and then electrosprayed directly into LCQ-Deca mass spectrometer. The MS spray voltage was maintained at 3.2 kV and the capillary temperature was at 200 °C. Ion trap collision energy of MS/MS was 35% and ions were collected covering the mass range from m/z 400 to 2000.
Protein identification using MS/MS raw data was performed with SEQUEST software (University of Washington, licensed to Thermo Finnigan) based on the Arabidopsis database of NCBI (www.ncbi.nlm.nih.gov). A relative molecular mass of 57 (57 kDa) was added to the average molecular of cysteines in MS/MS data searching. Both b ions and y ions were included in the database search also. Protein identification results were filtered as described by Yu et al. (2000) with the correlation factor ( Xcorr, 1+ 
1.9, 2+ 2.2, 3+ 3.75) and the delta cross-correlation factor (DelCn 0.1).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksSupplementary dataReferences |
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Metabolic modification under clinostat rotation conditions
The characterization of metabolic changes is generally considered to be one of the most direct approaches for studying the physiological changes in plant cells subjected to clinostat rotation conditions (Obenland and Brown, 1994
; Vasilenko and Popova, 1996; Soga et al., 2001). In this study, Arabidopsis callus cells exposed for 8 h to H, or V treatment and S control showed no changes in growth (data not shown), but a reduction of glucose by 43.5% and of fructose by 37.6% in the H compared with the S-treated cells. By contrast, the starch content increased by 119% during the H treatment relative to S treatment (Fig. 1), while no differences was observed between the V and S specimens. The V-treatment samples, however, increased the amount of glucose and fructose 121% and 118%, respectively, compared with the S samples. This finding indicates that both vertical and horizontal rotation caused differences in carbohydrate metabolism of the callus cells, but that the two treatments led to different responses. To determine if these metabolic differences reflect simple changes in enzyme activity or changes in the types of proteins produced in response to these experimental conditions, the proteome in Arabidopsis callus cells treated by horizontal clinorotation have been compared with both vertical rotation and stationary growth type cells.
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Quantitative proteome alterations in Arabidopsis callus cells under clinostat rotation conditions
Proteins were extracted from Arabidopsis callus cells and separated by two-dimensional (2-D) gel electrophoresis. More than 2000 protein spots were detected reproducibly and about 1000 protein spots were quantified reliably in 2-D gel (Fig. 2). Comparisons were performed among 2-D gels of the samples treated by either the H or the V treatment or the S control. 80 protein spots were found with their volumes changed significantly (P <0.01) in H gels or V gels compared with S gels (Fig. 2; Table 1). According to their response to clinorotation (H or V), these 80 protein spots could be grouped into three categories on the basis of statistical analysis of their abundance in the 2-D gels (Table 1). The group I protein spots were increased or decreased in abundance and/or changed position only in the H samples, whereas no statistical relevant difference between the V and S samples were detected. Examples of group I proteins include protein spot no. 17 in Fig. 3A, B, and C, which is seen to increase only in the H samples and protein spot nos 11 and 14 in Fig. 4, which exhibit changes in both abundance and position under the H growth conditions (Fig. 4C, F). Group II-type changes in protein abundance were detected only in the V-treated samples. The protein spots no. 77 and 78 in Fig. 3D, E and F, which show a decrease in abundance only in the V samples (Fig. 3E), are typical of this group. The group III protein spots responded to both V and H types of rotations (see protein no. 36 in Fig. 3G, H, I). (More information about other changed proteins in these three groups can be found in the supplementary material that is available at JXB on-line.)
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Of the 80 differentially expressed protein spots identified in this study, 20 were of the group I type, in which changes in volume and/or position were only observed in the H samples. These group I proteins were excised from preparative gels for mass spectrometry analysis (Table 1). 18 protein spots were identified with high confidence using SEQUEST with MS/MS raw data (Table 2). Based on their functional properties, these proteins can be categorized as stress response proteins (the largest group), followed by proteins involved in metabolism, protein synthesis, cell wall formation, gene activate/transcription, and signaling.
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Stress responsiveness
The abundance of aldehyde dehydrogenase (ALDH2), glutathione-dependent formaldehyde dehydrogenase (GS-FDH), glutathione S-transferase (GST), and ornthine carbamoyltransferase (OCT) were found to be significantly increased in the H treatment relative to the V treatment samples (5.6, 3.5, 2.7, and 3.7-fold). By contrast, ornithine aminotransferase (OAT), basic chitinase, and ATP-AMP transphosphorylase were decreased about 3.2, 2.1, and 2.9-fold, respectively (Table 2). The elevated expression level of ALDH2 seen in this study (Table 2) is consistent with reported increased activities of two ALDHs from barley grown under drought and salt stress conditions (Ozturk et al., 2002
), and with increased transcripts of Arabidopsis callus cells subjected to the hyper-g treatment (Martzivanou and Hampp, 2003). The increases in GS-FDH and GST (Table 2), which participate in stress responses and detoxification (Bellocco et al., 2002; Tamura et al., 2002) were also consistent with the high transcript abundance in gravity and mechanically stimulated Arabidopsis root apex cells (Moseyko et al., 2002; Kimbrough et al., 2004).
The effects of altered gravity growth conditions on OCT and OAT is reported here for the first time. OCT catalyses the formation of L-citrulline from carbamoyl-p and L-ornithine (Orn), the first committed step in the biosynthesis of arginine (Bellocco et al., 2002; Tamura et al., 2002), while OAT is responsible for either the synthesis of proline (Yang and Kao, 1999; Lin et al., 2002) or the transformation of Orn into glutamate semialdehyde in young Arabidopsis plantlets (Roosens et al., 1998). Both of these two enzymes use Orn as a substrate, which is a precursor for glutamate, proline, polyamines, and alkaloids. OCT and OAT may play an important role in linking Orn to arginine and proline pools, respectively. It is assumed that the switch in protein levels between OCT and OAT in Arabidopsis calli in this study under H clinostat rotation might regulate the contributions of Orn to proline and polyamine pools. The abundance of these two enzymes for V and S were not statistically different (Table 1; spot nos 65 and 62), indicating clinostat motion itself does not affect significantly expression of these two proteins. The alternating expression of these enzymes under clinorotation in this study indicates that the effects of clinostat rotation may include a general stress response.
Enzymes of carbohydrates metabolism
In this study, it was observed that the fructokinase, fructose bisphosphate aldolase, mitochondrial NAD+-isocitrate dehydrogenase (NAD+-IDH), and NADP+-isocitrate dehydrogenase (NADP+-IDH) were up-regulated about 3.7, 2.6, 2.7, and 2.4-fold, respectively, in the H relative to the V treatment samples (Table 2). In plants, fructokinase and fructose bisphosphate aldolase play an important regulatory role in the flux of carbon through carbohydrate metabolism (Gonzali et al., 2001; Pego and Smeekens, 2000; Schaeffer et al., 1997). The cDNAs of fructokinase and fructose bisphosphate aldolase were reported up-regulated in Arabidopsis culture cells and root apex under alternating gravity conditions (Martzivanou and Hampp, 2003; Kimbrough et al., 2004). The up-regulation of fructokinase involved in fructose phosphorylation was correlated with a decrease in pool sizes of fructose in the H-treated cells in this study (Table 2; Fig. 1). Alterations in the accumulation of glucose and starch were also observed in the H compared to the V and the S samples (Fig. 1), but no key enzyme involved in glucose and starch metabolism was detected with a change in the amount of its protein under the same conditions. These results indicate the effect of clinostat rotation on the metabolism of fructose and glucose might be different.
The mitochondrial enzymes NAD+-IDH and NADP+-IDH play a critical role in regulating the tricarboxylic acid cycle (TAC) in plant cells (Behal and Oliver, 1998) and the pool size of NADH and NADPH, which are essential cofactors for many enzymatic reactions (Igamberdiev and Gardeström, 2003; Kim and Park, 2005). The ratio of NADH/NAD decreased in tobacco protoplasts under microgravity and was reported by Hampp et al. (1997). This is the first report of an effect of clinorotation on the mitochondrial enzymes NAD+-IDH and NADP+-IDH. Based on the central role of these enzymes in controlling carbohydrate cycling and in TAC, their increased abundance in the clinostat rotation-treated cells in the present study presumably reflect altered patterns of carbon and energy flux in response to clinostat rotation.
Clinorotation also caused an increase in the lipid-processing enzyme, stearoyl-acyl carrier protein (ACP) desaturase (Table 2). ACP desaturase can catalyse the desaturation of stearic acid to produce the mono-unsaturated oleic acid in the lipid biosynthesis pathway (Slocombe et al., 1994; Haralampidis et al., 1998). A study of the life cycle of Brassica rapa on board Mir in the Svet greenhouse indicated that the reserves of seeds developed in microgravity were stored preferentially in the form of starch rather than protein and lipid. The causes of this effect of microgravity on storage metabolism remain to be determined. The observed effect of clinostat rotation on ACP desaturase may be related to the other changes in the production and utilization of reserves in response to gravity changes.
Signalling
The 5.0-fold increase of protein disulphide isomerase (PDI) in the H samples (Table 2) suggests that altered gravity conditions also affect the functioning of the secretory apparatus. This enzyme is a protein thiol oxidoreductase and chaperone that catalyses the oxidation, reduction and isomerization of protein disulphides in the ER lumen (Li and Larkins, 1996). PDI has been shown to have an effect on many cellular functions including storage protein foldings, receptor activity, cell–cell interactions, gene expression, and actin filament polymerization (Noiva, 1999). To what extent the increase in PDI is related to enhanced rates of secretion or to the stress response remains to be determined.
Gene activation/transcription
Many studies of the transcriptional activation of gene expression have reported that transcription factors play a major role in stress response pathways (Busch et al., 2005; Henriksson and Nordin Henriksson, 2005). In this study, it has been found that the transcription initiation factor (TFIIF) ß subunit is up-regulated about 5.1-fold in the H treatment (Table 2). In Schizosaccharomyces pombe, one of the subunits of TFIIF (Tfg3) was reported to be involved in transcriptional regulation under stress conditions (e.g. high temperature: Kimura and Ishihama, 2004). The increase of TFIIF ß in Arabidopsis callus cells subjected to clinostat rotation suggests that TFIIF might also be involved in transcriptional regulation of the response of plant to gravitropic stimulation.
Protein synthesis
Protein synthesis in plant cells plays many important physiological roles in response to unfavourable conditions. The expressions of ribosomal protein genes, which play a crucial role in the synthesis of proteins, were observed to be enhanced under auxin stimulation and low temperature (Beltrán-Peña et al., 2002; Kim et al., 2004). In this study, a 34.1 kDa 60S acidic ribosomal protein with pI 4.93 and a 15.3 kDa 40S ribosomal protein with pI 5.62 were detected to be up-regulated about 4.0 and 2.0-fold, respectively, under H conditions (Table 2). The increase of ribosomal protein might enhance the translation process, or help ribosome functioning under clinostat conditions.
Cell wall biosynthesis
These data showed that two reversible glycosyl polypeptides (RGPs) (RGP-1: 40.7 kDa, pI 5.61; RGP-2: 40.9 kDa, pI 5.76) are down-regulated about 3.6 and 3.7-fold, respectively, in the H-reatment samples relative to the V-treatment samples (Table 2). RGPs, which may be involved in plant cell wall synthesis, are found in Golgi fractions and have been shown to bind UDP-glucose, UDP-xylose, and UDP-galactose in a reversible manner, with substrate specificity for both nucleotides and sugar (Zhao and Liu, 2001). Changes in cell wall properties of plants grown under space microgravity and altered gravity conditions have been reported by others (Skagen and Iversen, 2000; Soga et al., 2001). The altered expression of RGPs in the H-treated cells observed in this study suggests that the metabolism involved in cell wall biosynthesis might be modified under clinostat rotation conditions.
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Concluding remarks |
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TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksSupplementary dataReferences |
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In this study, a systematic proteomic analysis of the proteins in cultured Arabidopsis cells grown on a horizontally rotating clinostat (simulated weightlessness) is reported. The 18 identified proteins that responded to this treatment are involved in a wide range of cellular processes, including general stress responses, general metabolism, gene activation/transcription, protein synthesis, and cell wall biosynthesis. Several proteins, such as OCT and OAT, were not known previously to be involved in responding to the clinostat rotation stimulus. Although OCT and OAT have not been reported to play a role in gravitropism, these results imply involvement of the ornithine synthesis pathway of Arabidopsis calli in the response to constant omnilateral gravity stimulation. In addition, a stress response transcription initiation factor (TFIIF) was found to be enhanced by clinostat rotation. These results provide useful molecular information at the protein level in order to understand the effect of the randomized orientation of the gravity vector on plants at the cellular level.
The proteins analysed in this study represent only a small fraction of the Arabidopsis proteome. Many other gravity responsive proteins still remain to be identified. These include many low abundance proteins, membrane proteins, and nuclear proteins, all of which are believed to play key roles in cellular organization, cell wall modification, and stress signal transduction. In addition, different types of gravity stimulation, dynamically (clinostat rotation) and statically (centrifugation) has been reported to result in different gene expression (Martzivanou and Hampp, 2003; Centis-Aubay et al., 2003; Yoshioka et al., 2003), similar alterations should occur at protein levels. Deeper proteomic analysis at plant cellular and organ levels in different gravitational force environments, such as hypergravity and microgravity, will hopefully provide further insight into the effects of gravity on plant growth and development.
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Supplementary data |
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Additional detailed information about the protein spots that showed significantly changed intensity in the clinostat rotation-treated cells is available at JXB on-line.
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Acknowledgements |
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The authors are indebted to Professor Andrew Staehelin and Professor Zhangcheng Tang for helpful suggestions and comments on the manuscript. This work was supported by a grant from the Chinese Academy of Sciences (Grant No. KSCX2-SW-322).
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References |
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