A Proteomic Approach to Analysing Responses of Arabidopsis Thaliana Callus Cells to Clinostat Rotation

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Journal of Experimental Botany, Vol. 57, No. 4, pp. 827–835, 2006 doi:10.1093/jxb/erj066 Advance Access publication 31 January, 2006 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

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

Hui Wang1, Hui Qiong Zheng1,*, Wei Sha2, Rong Zeng2 and Qi Chang Xia2 1 Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China 2

Research Center for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200032, China

Received 2 August 2005; Accepted 18 November 2005

Abstract that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins Callus cells of Arabidopsis thaliana (cv. Landsberg involved in general stress responses, metabolic path- erecta) were exposed for 8 h to a horizontal clinostat ways, gene activation/transcription, protein synthesis, rotation (H, simulated weightlessness), a vertical clino- and cell wall biosynthesis. stat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and Key words: Arabidopsis thaliana, callus cells, clinostat rotation, fructose apparently decreased, while starch content proteomics. 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 Introduction rotation have been comparatively analysed by high- Gravity has a profound influence on plant growth and resolution two-dimensional (2-D) gel electrophoresis development. Removal of the influence of gravitational and mass spectrometry. Image analysis of silver- acceleration by spaceflight causes a wide range of cellular stained 2-D gels revealed that 80 protein spots showed changes in plants (Cogoli and Gmu¨nder, 1991; Hampp quantitative and qualitative variations that were signifi- et al., 1997). Since the opportunity to study the role of cantly (P <0.01) and reproducibly different between the gravity in space is limited, clinostats have been developed clinorotated (H or V) and the stationary control samples. to partially simulate, in ground-based experiments, the Protein spots excised from 2-D gels were analysed by weightless environment of space. Growing plants on the microbe high performance liquid chromatography-ion clinostat with a horizontal axis corresponds to constant trap-mass spectrometry (LC-IT-MS) to obtain the tandem omnilateral gravity stimulation (Brown et al., 1976; Smith mass (MS/MS) spectra. 18 protein spots, which showed et al., 1997). Whole seedlings, germinated and grown on significant expression alteration only under the H con- clinostats, show an absence of certain features that are dition compared with those under V and S conditions, characteristic of gravitropically-grown plants (Shen-Miller were identified. Of these proteins, seven were involved and Gordon, 1967; Stankovic´ et al., 2001). At the cellular in stress responses, and four protein spots were iden- level, clinostat treatments produce specific effects on plant tified as key enzymes in carbohydrate metabolism cells, such as alterations in cell wall composition, increased and lipid biosynthesis. Two reversibly glycosylated cell production of heat-soluble proteins, and changes in cellular wall proteins were down-regulated in the H samples. energy metabolism (reviewed by Claassen and Spooner, Other proteins such as protein disulphide isomerase, 1994; Shen-Miller and Hinchman, 1995; Vasilenko and transcription initiation factor IIF, and two ribosomal Popova, 1996). Other changes include a more random proteins also exhibited altered expression under the H distribution of plastids (amyloplasts) and an increase in condition. The data presented in this study illustrate the number and volume of nucleoli (Shen-Miller and

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. The online version of this article has been published under an Open Access model. Users are entitled to use, reproduce, disseminate, or display the Open Access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Society for Experimental Biology are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact: [email protected] 828 Wang et al. Hinchman, 1995; Smith et al., 1997). Protoplasts of at a radius of 2.5 cm). In addition to the stationary 1 g control (S), Brassica napus exposed to both microgravity and clinostat a vertically oriented clinostat (V) rotating at 10 rpm was used rotation exhibited a decrease in the cell divisions and as a control to evaluate potential mechano-stimulation artefacts from the clinostat motor and the side-effects of rotation itself. For abnormal microtubule arrays (Skagen and Iversen, 2000). each experiment, three calli (fresh weight 80–100 mg each) were A number of recent studies have shown that the exposure grown at the same distance (2.5 cm) to a central dot on Petri dishes, of Arabidopsis seedlings and callus cells to gravity and the Petri dishes were mounted on the clinostat as described by stimulation, hyper g-forces or clinostat rotation induces Liu et al. (1993). alterations in gene expression (Moseyko et al., 2002; Carbohydrate analysis Centis-Aubay et al., 2003; Martzivanou and Hampp, About 10 mg of the callus cells were incubated in 80% ethanol at 2003; Yoshioka et al., 2003; Kimbrough et al., 2004). 60 8C for 1 h, dried, and extracted in 200 ll ddH2O at room tem- These studies confirmed the findings of several earlier perature for 15 min with occasional vortexes, followed by centri- studies that showed that altered gravity conditions led to fugation at 10 000 g for 10 min. The supernatants were used for the synthesis of new proteins in the root tips of corn and soluble sugar assays according to Hampp et al. (1994). The ab- Arabidopsis seedlings (Feldman, 1983; Feldman and sorbance was recorded on a microplate reader system (Sunrise Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 Tecan, USA). The starch of the pellets was determined as previously Gildow, 1984; Friedmann and Poovaiah, 1991). However, described by Obenland and Brown (1994). Five independent the findings of Rasmussen et al. (1992), who reported samples were measured to obtain the mean values 6SE. a major decrease of total protein content and the loss of distinct bands from the electrophoresis pattern in rapeseed Protein extraction and 2-DE analysis callus grown in orbit, should not be overlooked. 1 g of calli were ground in liquid nitrogen to a fine powder and Proteome analysis provides a means for analysing resuspended in an ice-cold solution of 10% w/v trichloroacetic acid differential gene expression at the protein levels, and this (TCA) in acetone with 0.07% w/v DTT for at least 1 h at ÿ20 8C, and centrifuged for 30 min at 35 000 g. The pellets were rinsed twice with approach has proved to be a powerful tool for analysing the acetone containing 0.07% w/v DTT for 1 h at ÿ20 8C and then responses of plants to environmental stresses, including lyophilized. The resulting powder pellet was solubilized in lysis drought, salt, and high and low temperatures (de Vienne buffer (7 mol lÿ1 urea, 2 mol lÿ1 thiourea, 4% CA-630, 32 mmol lÿ1 ÿ1 ÿ1 et al., 1999; Salekdeh et al., 2002; Bae et al., 2003; Majoul TRIS-HCl pH 6.8, 1 mmol l PMSF, 14 mmol l DTT, and 0.2% et al., 2004; Yan et al., 2005). The current study extends Triton X-100) for 1 h at 17 8C and then centrifuged at 12 000 g for 15 min. The proteins in the supernatant were precipitated by adding this type of analysis to plants subjected to altered gravity four volumes of ice-cold acetone, incubated at ÿ20 8C for at least conditions. To this end, the proteomes of Arabidopsis 2 h, and centrifuged at 12 000 g for 15 min. The pellets were callus cells exposed to horizontal clinostat rotation (H), dissolved in rehydration buffer containing 8 mol lÿ1 urea, 2% w/v ÿ1 vertical clinostat rotation (V) and stationary control (S) CHAPS, 18 mmol l DTT, 0.5% w/v IPG buffer pH 4–7 and a trace growth conditions have been characterized. The functional of bromophenol blue. The protein concentrations were quantified using the Bradford method (Bradford, 1976). Three samples per implications of the observed changes in protein expression treatment (H, V and S) were prepared from individual cell cultures. in response to clinostat rotation are discussed. All samples were stored at ÿ80 8C prior to electrophoresis. 2-DE was performed primarily according to Yu et al. (2000). One- dimensional isoelectric focusing (IEF) was performed on an immo- Materials and methods bilized pH gradient strip (IPG strip; pH4-7, linear, 13 cm, Pharmacia). 100 lg and 800 lg of total proteins were loaded onto analytical and Plant materials and cell culture conditions preparative gels, respectively. IEF was conducted in three steps: 500 Callus cultures were produced from Arabidopsis thaliana (cv. V for 1 h, 1000 V for 1 h, and 8000 V for 6 h. Focused strips were Landsberg erecta) seeds in MS medium (Murashige and Skoog, equilibrated twice for 15 min in 2 ml equilibration solution. The first 1962) supplemented with 3% sugar and 2 mg lÿ1 2,4-D. The sus- equilibration step was performed in a solution containing 6 mol lÿ1 pension cultures were grown in 250 ml conical flasks in the dark at urea, 30% glycerol, 2% SDS, 2% DTT, 50 mmol lÿ1 TRIS-HCl 22–25 8C in an orbital shaker (130 rpm) and were subcultured buffer (pH 8.8). The second equilibration step was performed in every week. The suspension cultures were taken as stock for repeated a solution modified by the replacement of DTT by 2.5% iodoaceta- callus formation. At 6–7 d after subculture, the cultures were trans- mide. Two-dimensional SDS-PAGE (2-DE) was performed with ferred to solid medium supplemented with 1% agar (as described 1 mm thick, 12.5% T SDS- polyacrylamide gel (Hofer SE 600 in by Martzivanou and Hampp, 2003). Calli with a diameter of about a vertical slab). The gels were run at 10 mA per gel for the first 30 min 1 mm were used for experiments after 1 week of growth on Petri dishes. and followed by 25 mA per gel. After 2-DE, the analytical gels were stained with ammoniacal silver nitrate according to the procedure Clinostat treatments described by Yu et al. (2001), and the preparative gels were stained A one-axis 1-p clinostat facility (SB-01) was used to simulate with colloidal Coomassie Brilliant blue G-250 (Bradford, 1976). At weightlessness. Characteristic physical details of this instrument were least three replicates were performed for each sample. given by Liu et al. (1993). Briefly, this device was designed as two groups of orthogonal axes allowing simultaneous rotation around Image acquisition and analysis each of four horizontal and vertical axes. The rotation rate of the The silver-stained 2-D gels were scanned using a GS710 imaging clinostat (0.5–200 rpm) could be adjusted and controlled by a control densitometer (Bio-Rad) in transmissive mode. Spot detection, box. The clinostat rotation treatment in this study consisted of a quantification, and matching were performed using PDQuest 7.1 clinostat oriented horizontally (H) and rotating at 10 rpm (e.g. the software (Bio-Rad). A matchset consisting of 15 images, five for level of acceleration applied to the materials was less than 2.8310ÿ3 g the H samples, five for the V samples, and five for the S samples was Response of callus calls to clinostat rotation 829 created, and one image from S was selected as the matchset standard (data not shown), but a reduction of glucose by 43.5% and for spot matching. The match rate of every gel to the selected standard of fructose by 37.6% in the H compared with the S-treated gel was higher than 90% according to the analytical results by cells. By contrast, the starch content increased by 119% PDquest 7.1 software. Only those with significant and reproducible changes were considered to be differentially accumulated proteins. during the H treatment relative to S treatment (Fig. 1), while The abundance of each protein spot was estimated by the percentage no differences was observed between the V and S speci- volume, for example, the individual spot volumes were normalized mens. The V-treatment samples, however, increased the by dividing their optical density (OD) values by the total OD values amount of glucose and fructose 121% and 118%, respect- of all the spots present in the gel, and expressed as % Vol. The ively, compared with the S samples. This finding indicates significance of expression differences of protein spots between clinostat rotational conditions (H or V) and stationary control (S) that both vertical and horizontal rotation caused differences was estimated by Student’s t-test, P <0.01. in carbohydrate metabolism of the callus cells, but that the two treatments led to different responses. To deter- In-gel digestion mine if these metabolic differences reflect simple changes Protein spots were excised from the preparative gels, rinsed with water in enzyme activity or changes in the types of proteins ÿ1 Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 (Milli-Q) three times, destained twice with 25 mmol l NH4HCO3 produced in response to these experimental conditions, in 50% acetonitrile (ACN), reduced with 100 mmol lÿ1 DTT in ÿ1 ÿ1 the proteome in Arabidopsis callus cells treated by hori- 100 mmol l NH4HCO3, alkylated with 200 mmol l iodoacetamide ÿ1 zontal clinorotation have been compared with both ver- in 100 mmol l NH4HCO3, dried by lyophilization, and digested ÿ1 ÿ1 for 20 h at 37 8C with 5 ng ll trypsin in 25 mmol l NH4HCO3, tical rotation and stationary growth type cells. pH 8.3. The peptides were extracted three times with 0.1% trifluoro- acetic acid (TFA) in 60% ACN. The supernatants were pooled and Quantitative proteome alterations in Arabidopsis callus lyophilized for mass spectrometric analysis. cells under clinostat rotation conditions Proteins were extracted from Arabidopsis callus cells and LC-IT-MS and protein identification separated by two-dimensional (2-D) gel electrophoresis. In-gel digested samples were re-dissolved in 0.1% v/v TFA and desalted using a C18 Zip Tip (Millipore). The separation and iden- More than 2000 protein spots were detected reproducibly tification of the peptide mixtures was conducted by a Finnigan LCQ and about 1000 protein spots were quantified reliably in Deca XP ion-trap micro-electrospray mass spectrometer (Thermo- 2-D gel (Fig. 2). Comparisons were performed among 2-D Quest, San Jose, CA, USA) coupled with a reversed-phase high- gels of the samples treated by either the H or the V treat- performance liquid chromatography (HPLC) system (ThermoQuest). ment or the S control. 80 protein spots were found with For LC separation, a 0.153120 mm column (RP-C18, ThermoHy- persil, San Jose, CA, USA) was used. Solvent A was 0.1% v/v formic their volumes changed significantly (P <0.01) in H gels or acid and solvent B was 0.1% v/v formic acid in 100% v/v ACN. The V gels compared with S gels (Fig. 2; Table 1). According to gradient was held at 2% v/v solvent B for 15 min, and increased their response to clinorotation (H or V), these 80 protein linearly to 98% v/v solvent B in 90 min. The peptides were eluted spots could be grouped into three categories on the basis of from a C18 microcapillary column at a flow rate of 150 ll per min and statistical analysis of their abundance in the 2-D gels then electrosprayed directly into LCQ-Deca mass spectrometer. The MS spray voltage was maintained at 3.2 kV and the capillary tempera- (Table 1). The group I protein spots were increased or de- ture was at 200 8C. Ion trap collision energy of MS/MS was 35% and creased in abundance and/or changed position only in the H ions were collected covering the mass range from m/z 400 to 2000. samples, whereas no statistical relevant difference between Protein identification using MS/MS raw data was performed with the V and S samples were detected. Examples of group I SEQUEST software (University of Washington, licensed to Thermo proteins include protein spot no. 17 in Fig. 3A, B, and C, Finnigan) based on the Arabidopsis database of NCBI (www.ncbi. nlm.nih.gov). A relative molecular mass of 57 (57 kDa) was added which is seen to increase only in the H samples and protein to the average molecular of cysteines in MS/MS data searching. spot nos 11 and 14 in Fig. 4, which exhibit changes in both Both b ions and y ions were included in the database search also. abundance and position under the H growth conditions Protein identification results were filtered as described by Yu et al. (Fig. 4C, F). Group II-type changes in protein abundance (2000) with the correlation factor ( Xcorr, 1+ > 1.9, 2+ >2.2, 3+ were detected only in the V-treated samples. The protein > > 3.75) and the delta cross-correlation factor (DelCn 0.1). spots no. 77 and 78 in Fig. 3D, E and F, which show a decrease in abundance only in the V samples (Fig. 3E), Results and discussion are typical of this group. The group III protein spots responded to both V and H types of rotations (see protein Metabolic modification under clinostat rotation no. 36 in Fig. 3G, H, I). (More information about other conditions changed proteins in these three groups can be found in the The characterization of metabolic changes is generally supplementary material that is available at JXB on-line.) considered to be one of the most direct approaches for Of the 80 differentially expressed protein spots identified studying the physiological changes in plant cells subjected in this study, 20 were of the group I type, in which changes to clinostat rotation conditions (Obenland and Brown, in volume and/or position were only observed in the H 1994; Vasilenko and Popova, 1996; Soga et al., 2001). In samples. These group I proteins were excised from pre- this study, Arabidopsis callus cells exposed for 8 h to H, or parative gels for mass spectrometry analysis (Table 1). 18 V treatment and S control showed no changes in growth protein spots were identified with high confidence using 830 Wang et al.

160 SEQUEST with MS/MS raw data (Table 2). Based on their glucose functional properties, these proteins can be categorized frutrose 140 as stress response proteins (the largest group), followed starch by proteins involved in metabolism, protein synthesis, cell wall formation, gene activate/transcription, and signaling. 120

Stress responsiveness 100 The abundance of aldehyde dehydrogenase (ALDH2), glutathione-dependent formaldehyde dehydrogenase (GS- 80 FDH), glutathione S-transferase (GST), and ornthine carbamoyltransferase (OCT) were found to be signiﬁcantly 60 increased in the H treatment relative to the V treatment relative of control (%)

samples (5.6, 3.5, 2.7, and 3.7-fold). By contrast, ornithine Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 40 aminotransferase (OAT), basic chitinase, and ATP-AMP transphosphorylase were decreased about 3.2, 2.1, and 2.9- 20 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 0 HVgrown under drought and salt stress conditions (Ozturk Fig. 1. Differences in sugar and starch concentrations in Arabidopsis et al., 2002), and with increased transcripts of Arabidopsis culture cells subjected to either horizontal or vertical clinorotation callus cells subjected to the hyper-g treatment (Martzivanou compared with control cells grown under stationary conditions. All treatments were for a period of 8 h. The data represent the mean of ﬁve and Hampp, 2003). The increases in GS-FDH and GST replications. Bars designate standard error. S: stationary, H: horizontal (Table 2), which participate in stress responses and de- clinorotation, V: vertical clinorotation. toxiﬁcation (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).

Fig. 2. Images of 2-D gels of proteins extracted from Arabidopsis calli exposed to clinostat rotation. Proteins were visualized by silver staining. The arrows indicate 80 protein spots that changed in abundance and/or position in the horizontal and/or vertical clinorotation samples. The background gel was of a stationary control (the 80 protein spots were displayed on two gels to increase the spatial resolution of the spots). The ranges of pI and molecular masses (kDa) are indicated. Response of callus calls to clinostat rotation 831 Table 1. Distribution of protein spots whose intensities were altered by 8 h of horizontal and vertical clinorotation treatment in Arabidopsis callus cells

Spot. noa Obs. Mr. Treatmentc Spot. no. Obs. Mr. Treatment (kDa)/pIb (kDa)/pI HV HV

Group I Group III 11 41/5.87 +/p + 1 65/5.30 + + 13 34/5.15 + NC 2 61/5.68 – – 14 35/6.09 +/p + 3 52/4.65 – – 16 32/6.37 + NC 4 56/5.70 + + 17 30/6.05 + NC 5 54/5.89 – – 19 31/5.58 – NC 7 50/5.14 – – 20 31/5.49 – NC 9 48/5.13 – –

21 26/4.82 + NC 10 44/5.44 – – Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 26 23/5.41 + NC 15 30/6.89 + + 31 21/4.73 ND NC 22 25/5.01 + + 32 18/4.62 ND NC 23 27/5.16 ÿÿ 35 15/5.35 + NC 24 27/5.57 – – 40 25/6.05 – NC 25 23/5.27 + + 60 36/5.76 + NC 27 20/5.20 + + 62 36/5.92 – NC 29 24/6.38 ND – 63 30/5.13 + NC 33 18/4.66 + + 64 33/5.43 + NC 36 14/5.34 + + Fig. 3. Details of silver-stained 2-D gels showing examples of protein 65 30/5.78 + NC 37 35/4.13 – – spots whose abundance changed (arrows) after exposure to either vertical 67 29/6.91 – NC 39 26/6.02 + + clinorotation (B, E, H) or horizontal clinorotation (C, F, I) compared with 75 27/5.79 + NC 41 55/5.58 + + those that remained stationary (A, D, G). Spot numbers correspond to Group II 42 55/5.65 + + those shown in the reference gel (Fig. 2) and Table 1. 6 46/4.76 NC – 44 63/5.85 + + 8 49/5.19 NC N 45 63/5.91 ND – 12 35/5.26 NC – 46 49/5.28 + + 18 30/6.52 NC + 47 5/5.33 – – 28 24/5.73 NC + 48 51/5.42 – – 30 20/5.38 NC – 49 53/5.73 + + 34 15/4.99 NC – 50 50/5.62 – – 38 16/4.59 NC – 51 46/5.46 + + 43 65/5.86 NC N 52 52/5.85 + + 53 47/5.86 NC + 56 50/6.15 + + 54 52/5.90 NC + 57 46/6.62 – ND 55 53/6.00 NC + 58 46/6.73 – – 59 37/5.76 NC – 61 38/5.80 + + 66 30/6.95 NC + 68 29/7.11 – – 70 29/4.47 NC – 69 26/4.49 + + 71 27/4.42 NC – 73 24/5.80 – – 72 26/5.40 NC – 76 17/5.06 – – 74 29/6.06 NC + 79 15/5.79 – – 77 21/5.81 NC – 80 32/6.44 + + 78 20/5.79 NC – Fig. 4. Details of silver-stained 2-D gels of proteins whose position a Spot no is as the index in the reference gel. changed (arrows) after exposure to the H rotation treatment (C, F) b Obs., observed molecular weights and isoelectric points, which were compared with the V rotation treatment (B, E) and the stationary control estimated from electrophoretic mobilities. (A, D). Arrows in top panel: Aldehyde dehydrogenase (ALDH2, spot c +, Up-regulated under rotation condition; +/p: up-regulated in no.11). Arrows in bottom panel: Glutathione-dependent formaldehyde abundance and changed in position; –, down-regulated; NC, no change, dehydrogenase (spot no.14). N, detected only after rotation treatment; ND, not determined. H, horizontal clinorotation, V, vertical clinorotation.

of these two enzymes use Orn as a substrate, which is a The effects of altered gravity growth conditions on OCT precursor for glutamate, proline, polyamines, and alkaloids. and OAT is reported here for the first time. OCT catalyses OCT and OAT may play an important role in linking Orn the formation of L-citrulline from carbamoyl-p and L- to arginine and proline pools, respectively. It is assumed ornithine (Orn), the first committed step in the biosynthesis that the switch in protein levels between OCT and OAT of arginine (Bellocco et al., 2002; Tamura et al., 2002), in Arabidopsis calli in this study under H clinostat rota- while OAT is responsible for either the synthesis of tion might regulate the contributions of Orn to proline and proline (Yang and Kao, 1999; Lin et al., 2002) or polyamine pools. The abundance of these two enzymes the transformation of Orn into glutamate semialdehyde in for V and S were not statistically different (Table 1; spot young Arabidopsis plantlets (Roosens et al., 1998). Both nos 65 and 62), indicating clinostat motion itself does not 832 Wang et al. Table 2. Identification results of proteins with altered expression in Arabidopsis thaliana callus cells subjected to horizontal clinorotation compared to that under vertical clinorotation using MS/MS raw data

Spot noa Mr (kDa)/pIb Peptides Coveraged Relative volume (%)e Identity/accession number f identiﬁedc HV

Stress 11 58.6/7.1 87/22 43.49% 0.23160.030 0.04160.005 Aldehyde dehydrogenase (ALDH2), A. thaliana [NP_190383.1] 14 40.6/6.75 21/8 30.34% 0.12960.011 0.03760.007 Glutathione-dependent formaldehyde dehydrogenase, A. thaliana [AAB06322.1] 26 24.2/5.49 24/10 41.8% 0.09060.005 0.03960.005 Glutathione S-transferase ERD-13, A. thaliana [P42761] 65 41.2/8.46 9/4 14.40% 0.04460.002 0.01260.001 Ornithine carbamoyltransferase, A. thaliana [CAA04115.1] 62 52.2/7.15 9/5 13.05% 0.02360.005 0.07460.009 Onithine aminotransferase, A. thaliana [NP_199430.1] 67 36.2/7.81 54/8 42.09% 0.03760.003 0.07860.012 Basic chitinase, A. thaliana [AAG51023.1] Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 40 26.9/6.91 25/13 44.72% 0.12260.013 0.35560.022 ATP-AMP transphosphorylase, A. thaliana [O82514] Metabolism 17 40.6/6.71 12/7 25.40% 0.12260.016 0.04660.005 NAD+ isocitrate dehydrogenase, A. thaliana [AAM60999.1] 60 45.7/6.13 9/6 20.0% 0.12260.012 0.05060.005 NADP+ isocitrate dehydrogenase, A. thaliana [AAM13090.1] 64 38.5/6.05 9/6 25.14% 0.10060.012 0.03860.003 fructose bisphosphate aldolase, A. thaliana [NP_190861.1] 63 35.2/5.3 32/14 50.46% 0.19960.009 0.05460.006 Fructokinase, A. thaliana [AAM91113.1] 13 45.2/6.29 8/5 19.75% 0.31260.034 0.09960.011 Stearoyl-acyle-carrier protein desaturase, A. thaliana [AAM65642.1] Signalling 16 39.5/5.8 10/5 17.73% 0.33260.041 0.06760.013 protein disulphide isomerase A6 precursor, [O22263] Gene activation/transcription 75 29.7/6.47 4/2 13.03% 0.09760.010 0.01960.004 Transcription initiation factor II F b subunit family protein, A. thaliana [NP_177683.1] Protein synthesis 21 34.1/4.93 4/3 16.56% 0.17060.012 0.04360.007 Putative 60S acidic ribosomal protein, 59partial, A. thaliana [AAD56335.1] 35 15.3/5.62 16/7 47.92% 0.28760.025 0.14160.040 40S ribosomal protein S12-3, A. thaliana [Q9SKZ3] Cell wall biosynthesis 19 40.7/5.61 28/16 48.74% 0.06360.009 0.22760.031 Reversibly glycosylated polypeptide-1, A. thaliana [NP_186872.1] 20 40.9/5.76 34/17 51.39% 0.04160.006 0.15160.020 Reversibly glycosylated polypeptide-2, A. thaliana [NP_197069.1] a Index in the reference gel. b Expected Mr and pI which were calculated on the complete sequences. c Peptides had been identified: the number before the solidus represents the amount of peptides identified containing overlaps and the number behind the solidus represents the amount of peptides excluding overlaps. d The mass of peptides identified here as a percentage of the total mass of the protein. e The individual spot volumes were expressed as a percentage of the total volume in all of the spots present in the gel. H: cells exposed to the horizontal clinorotation; V: cells under vertical clinorotation were used as control for the effect of rotation alone. f Accession number in NCBI, SWISS-Prot, or PIR.

affect significantly expression of these two proteins. The al- regulatory role in the flux of carbon through carbohydrate ternating expression of these enzymes under clinorotation metabolism (Gonzali et al., 2001; Pego and Smeekens, in this study indicates that the effects of clinostat rotation 2000; Schaeffer et al., 1997). The cDNAs of fructokinase may include a general stress response. and fructose bisphosphate aldolase were reported up- regulated in Arabidopsis culture cells and root apex under Enzymes of carbohydrates metabolism alternating gravity conditions (Martzivanou and Hampp, In this study, it was observed that the fructokinase, fructose 2003; Kimbrough et al., 2004). The up-regulation of bisphosphate aldolase, mitochondrial NAD+-isocitrate de- fructokinase involved in fructose phosphorylation was hydrogenase (NAD+-IDH), and NADP+-isocitrate dehy- correlated with a decrease in pool sizes of fructose in the drogenase (NADP+-IDH) were up-regulated about 3.7, H-treated cells in this study (Table 2; Fig. 1). Alterations in 2.6, 2.7, and 2.4-fold, respectively, in the H relative to the accumulation of glucose and starch were also observed the V treatment samples (Table 2). In plants, fructokinase in the H compared to the V and the S samples (Fig. 1), but and fructose bisphosphate aldolase play an important no key enzyme involved in glucose and starch metabolism Response of callus calls to clinostat rotation 833 was detected with a change in the amount of its protein study, it has been found that the transcription initiation under the same conditions. These results indicate the effect factor (TFIIF) b subunit is up-regulated about 5.1-fold in of clinostat rotation on the metabolism of fructose and the H treatment (Table 2). In Schizosaccharomyces pombe, glucose might be different. one of the subunits of TFIIF (Tfg3) was reported to be in- The mitochondrial enzymes NAD+-IDH and NADP+- volved in transcriptional regulation under stress conditions IDH play a critical role in regulating the tricarboxylic acid (e.g. high temperature: Kimura and Ishihama, 2004). The cycle (TAC) in plant cells (Behal and Oliver, 1998) and the increase of TFIIF b in Arabidopsis callus cells subjected pool size of NADH and NADPH, which are essential to clinostat rotation suggests that TFIIF might also be cofactors for many enzymatic reactions (Igamberdiev and involved in transcriptional regulation of the response of Gardestro¨m, 2003; Kim and Park, 2005). The ratio of plant to gravitropic stimulation. NADH/NAD decreased in tobacco protoplasts under microgravity and was reported by Hampp et al. (1997). Protein synthesis This is the first report of an effect of clinorotation on the

Protein synthesis in plant cells plays many important Downloaded from https://academic.oup.com/jxb/article/57/4/827/558852 by guest on 01 October 2021 + + mitochondrial enzymes NAD -IDH and NADP -IDH. physiological roles in response to unfavourable conditions. Based on the central role of these enzymes in controlling The expressions of ribosomal protein genes, which play a carbohydrate cycling and in TAC, their increased abun- crucial role in the synthesis of proteins, were observed to be dance in the clinostat rotation-treated cells in the present enhanced under auxin stimulation and low temperature study presumably reflect altered patterns of carbon and (Beltrań-Penã et al., 2002; Kim et al., 2004). In this study, energy flux in response to clinostat rotation. a 34.1 kDa 60S acidic ribosomal protein with pI 4.93 and Clinorotation also caused an increase in the lipid- a 15.3 kDa 40S ribosomal protein with pI 5.62 were processing enzyme, stearoyl-acyl carrier protein (ACP) detected to be up-regulated about 4.0 and 2.0-fold, re- desaturase (Table 2). ACP desaturase can catalyse the de- spectively, under H conditions (Table 2). The increase of saturation of stearic acid to produce the mono-unsaturated ribosomal protein might enhance the translation process, oleic acid in the lipid biosynthesis pathway (Slocombe or help ribosome functioning under clinostat conditions. et al., 1994; Haralampidis et al., 1998). A study of the life cycle of Brassica rapa on board Mir in the Svet green- Cell wall biosynthesis house indicated that the reserves of seeds developed in These data showed that two reversible glycosyl polypep- microgravity were stored preferentially in the form of tides (RGPs) (RGP-1: 40.7 kDa, pI 5.61; RGP-2: 40.9 kDa, starch rather than protein and lipid. The causes of this pI 5.76) are down-regulated about 3.6 and 3.7-fold, effect of microgravity on storage metabolism remain to respectively, in the H-reatment samples relative to the V- be determined. The observed effect of clinostat rotation treatment samples (Table 2). RGPs, which may be involved on ACP desaturase may be related to the other changes in plant cell wall synthesis, are found in Golgi fractions and in the production and utilization of reserves in response have been shown to bind UDP-glucose, UDP-xylose, and to gravity changes. UDP-galactose in a reversible manner, with substrate Signalling specificity for both nucleotides and sugar (Zhao and Liu, 2001). Changes in cell wall properties of plants grown The 5.0-fold increase of protein disulphide isomerase (PDI) under space microgravity and altered gravity conditions in the H samples (Table 2) suggests that altered gravity have been reported by others (Skagen and Iversen, 2000; conditions also affect the functioning of the secretory Soga et al., 2001). The altered expression of RGPs in apparatus. This enzyme is a protein thiol oxidoreductase the H-treated cells observed in this study suggests that and chaperone that catalyses the oxidation, reduction and the metabolism involved in cell wall biosynthesis might isomerization of protein disulphides in the ER lumen (Li be modified under clinostat rotation conditions. 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 ex- Concluding remarks pression, and actin filament polymerization (Noiva, 1999). To what extent the increase in PDI is related to enhanced In this study, a systematic proteomic analysis of the pro- rates of secretion or to the stress response remains to be teins in cultured Arabidopsis cells grown on a horizontally determined. rotating clinostat (simulated weightlessness) is reported. The 18 identified proteins that responded to this treatment Gene activation/transcription are involved in a wide range of cellular processes, in- Many studies of the transcriptional activation of gene cluding general stress responses, general metabolism, gene expression have reported that transcription factors play activation/transcription, protein synthesis, and cell wall a major role in stress response pathways (Busch et al., biosynthesis. Several proteins, such as OCT and OAT, were 2005; Henriksson and Nordin Henriksson, 2005). In this not known previously to be involved in responding to the 834 Wang et al. clinostat rotation stimulus. Although OCT and OAT have Bradford MM. 1976. A rapid and sensitive method for the not been reported to play a role in gravitropism, these quantitation of microgram quantities of protein using the principle results imply involvement of the ornithine synthesis of protein–dye binding. Analytical Biochemistry 72, 248–254. Brown AH, Dahl AO, Chapman DK. 1976. Limitation on the use pathway of Arabidopsis calli in the response to constant of the horizontal clinostat as a gravity compensator. Plant Physio- omnilateral gravity stimulation. In addition, a stress re- logy 58, 127–130. sponse transcription initiation factor (TFIIF) was found Busch W, Wunderlich M, Scho¨ffl F. 2005. Identification of novel to be enhanced by clinostat rotation. 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