Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress

Biochimica et Biophysica Acta 1834 (2013) 1969–1975

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Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress Chao Wang a,b, Chang Yi Wang a, Xue Qiang Zhao a, Rong Fu Chen a, Ping Lan a, Ren Fang Shen a,⁎ a b

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e

i n f o

Article history: Received 7 March 2013 Received in revised form 17 June 2013 Accepted 20 June 2013 Available online 2 July 2013 Keywords: 2-DE Aluminum Citrate Malate dehydrogenase Rhodotorula taiwanensis

a b s t r a c t Rhodotorula taiwanensis RS1 is a high-aluminum (Al)-tolerant yeast that can survive in Al concentrations up to 200 mM. The mechanisms for the high Al tolerance of R. taiwanensis RS1 are not well understood. To investigate the molecular mechanisms underlying Al tolerance and toxicity in R. taiwanensis RS1, Al toxicity-induced changes in the total soluble protein profile were analyzed using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. A total of 33 differentially expressed proteins responding to Al stress were identified from approximately 850 reproducibly detected proteins. Among them, the abundance of 29 proteins decreased and 4 increased. In the presence of 100 mM Al, the abundance of proteins involved in DNA transcription, protein translation, DNA defense, Golgi functions and glucose metabolism was decreased. By contrast, Al treatment led to increased abundance of malate dehydrogenase, which correlated with increased malate dehydrogenase activity and the accumulation of intracellular citrate, suggesting that Al-induced intracellular citrate could play an important role in detoxification of Al in R. taiwanensis RS1. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction In acidic soils (pH b 5), high concentrations of aluminum (Al) ions are very toxic to a variety of living organisms, including plants and microorganisms [1,2]. Recently, Al tolerance and toxicity in plants have been extensively studied. In general, plants are very sensitive to Al ions; micromolar concentrations of Al severely inhibit the root growth of most plant species [1,3]. Some microorganisms, however, can tolerate Al levels up to 100–200 mM [4–6]. Therefore, it is important to understand how these microorganisms tolerate such high Al levels. Rhodotorula sp. RS1 (identified as Rhodotorula taiwanensis RS1 [7]), a red yeast isolated from acidic soil, can survive in 200 mM Al [8]. Our previous study showed that the secretion of organic acids into the culture medium did not contribute to the high tolerance of RS1 to Al toxicity; however, the thickened cell wall in response to the high Al concentrations may play an important role in RS1 Al tolerance [8]. Although RS1 showed an extraordinary capability to tolerate Al, high levels of Al (N 100 mM) still significantly inhibited its growth [8]. The molecular mechanisms of toxicity and tolerance of RS1 to high Al remain unknown. Increasing evidence shows that changes in transcript levels do not always result in alterations in protein levels. Proteins are the direct executors of physiological functions, ultimately controlling the biological ⁎ Corresponding author. Tel.: +86 25 86881563; fax: +86 25 86881000. E-mail addresses: [email protected] (C. Wang), [email protected] (C.Y. Wang), [email protected] (X.Q. Zhao), [email protected] (R.F. Chen), [email protected] (P. Lan), [email protected] (R.F. Shen).

processes within cells [9]; therefore, it is crucial to investigate the changes in protein profiles of RS1 in response to Al stress, to identify the genetic and molecular components underlying high Al tolerance in RS1 cells. Such a study could also identify putative cellular targets of Al toxicity and potential tolerance mechanisms. Currently, twodimensional gel electrophoresis (2-DE) is the most frequently used method for studying global protein changes, and has been successfully employed to analyze protein changes and characterize marker proteins for specific physiological or stress responses of a cell or tissue [10–12]. Proteomic studies on Al stress in plants have been conducted [13–16]. However, there have been no proteomic analyses of microorganisms in response to Al stress. In the present study, we used 2-DE and mass spectrometry (MS) to identify proteins whose abundance change in response to high Al stress level in R. taiwanensis RS1. Bioinformatic tools were used to analyze the global changes of proteins and the functions of these proteins in RS1 cells under high Al stress, and the results could infer potential biomarkers for high Al tolerance and toxicity. 2. Materials and methods 2.1. Cell culture The yeast R. taiwanensis RS1 was deposited in the China General Microbiological Culture Collection Center (CGMCC) with the Accession No. CGMCC 2.4753, and maintained in glucose medium (GM) (per liter): glucose, 10 g; NaCl, 10 g; yeast extract, 0.5 g; peptone, 0.2 g; MgSO4·7H2O, 0.2 g; and agar, 10 g, pH 3.5, as described by Wang et al. [8]. For liquid culture, the strain was grown in GM medium without agar. The medium

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C. Wang et al. / Biochimica et Biophysica Acta 1834 (2013) 1969–1975

was autoclaved at 121 °C for 20 min followed by addition of an appropriate amount of filter-sterilized Al2(SO4)3 stock solution (pH 3.5) to the medium to obtain the required Al concentrations [8]. The strain cultured on solid GM was inoculated into the liquid medium without Al, and used as a seed culture. When the optical density at 600 nm (OD600) reached 0.6, yeast culture (1 mL) was added to 250 mL flasks containing 70 mL medium with 0 or 100 mM Al, and cultured on a shaker at 120 rpm for 24 h at 30 °C. Cells were collected by centrifugation at 3000 ×g for 5 min at 4 °C and washed with ice-cold 0.01 M EDTA to remove surface Al, according to the method of Wang et al. [8]. The washed cells were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent protein extraction, malate dehydrogenase (MDH) activity assay and estimation of intracellular malate and citrate content. 2.2. Protein extraction The frozen cells were thoroughly ground in a mortar in liquid nitrogen. The homogenate was dissolved in cell lysis buffer (50 mM Tris– HCl, pH 7.6, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and 1 mM dithiothreitol [DTT]) at 4 °C. The cell debris was removed by centrifugation at 2000 ×g for 10 min, and the supernatant was then centrifuged at 40000 ×g for 40 min at 4 °C. The proteins in the supernatant were precipitated using trichloroacetic acid/acetone method [17]. The final protein extracts were solubilized in hydration solution (7 M urea, 2 M thiourea and 4% [w/v] 3[(3-cholamidopropyl) dimethylammonio]1-propanesulphonate [CHAPS]) and protein concentrations were determined using the Bradford method [18].

entries, downloaded on February 21, 2012). The following search parameters were set in the Mascot software: taxonomic category, Saccharomyces cerevisiae; no MW/pI restrictions; enzyme, trypsin; missed cleavages, 1; mass tolerance, 100 ppm and the modifications of cysteine carbamidomethylation and methionine oxidation; significant protein MOWSE score at p b 0.05. The biological processes and molecular functions that involve the identified proteins were searched in the Saccharomyces Genome Database (http://www.yeastgenome.org/ and http:// mips.helmholtz-muenchen.de/genre/proj/yeast/). 2.5. MDH activity assay Frozen cells were ground in a mortar with liquid nitrogen. The homogenate was dissolved in potassium phosphate buffer (PBS, 100 mM, pH 7.5, 2 mM MgCl2 and 1 mM DTT). After the sample was centrifuged at 12000 ×g for 30 min at 4 °C, the supernatant was used for the determination of enzyme activity and protein concentrations. MDH activity was measured spectrophotometrically by monitoring the changes in absorbance at 340 nm, as described by Pines et al. [21] with slight modifications. The reaction mixture for the reductive MDH activity contained 100 mM PBS (pH 7.9), 0.15 mM NADH, 1 mM oxaloacetate, and a suitable enzyme solution. For oxidative MDH activity, the reaction mixture contained 100 mM PBS (pH 7.9), 10 mM L-malate, 1 mM NAD+ and an aliquot of enzyme solution. Enzyme activity was expressed as micromoles NADH oxidized/NAD+ reduced per minute of reaction time per milligram protein. 2.6. Estimation of intracellular malate and citrate content

2.3. 2-DE 2-DE was carried out according to the methods of Li et al. [19], with slight modifications. The solubilized protein extract was diluted in rehydration buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 0.2% immobilized pH gradient [IPG] buffer [pH 4–7, GE Healthcare, USA], 40 mM DTT and 0.01% [w/v] bromophenol blue), then centrifuged at 10000 ×g for 5 min at room temperature. For each replicate, 800 μg of total proteins was loaded into a 17 cm long and pH 4–7 IPG strip (Bio-Rad, USA), and rehydrated for 16 h at room temperature. Isoelectric focusing was performed at 17 °C for a total of 70000 Vh. Focused strips were equilibrated in 10 mL of equilibration solution (50 mM Tris–HCl, pH 6.8, 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS and 2% [w/v] DTT) for 15 min, and then for 15 min in 10 mL of alkylating solution (50 mM Tris–HCl, pH 6.8, 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS and 2.5% [w/v] iodoacetamide). The IPG strips were transferred to a 12.5% SDS-PAGE gel to separate the proteins according to their molecular weight (MW). The gels were stained using Coomassie brilliant blue R250. Gel images were scanned using Gel Doc™ XR+ (Bio-Rad) at a resolution of 300 dpi and analyzed using PDQuest 8.01 (Bio-Rad) for spot detection, quantification, and matching, as well as comparative and statistical analyses. 2.4. Protein identification Proteins were identified according to the method described by Wu et al. [20]. In brief, the protein spots were excised, and dehydrated in acetonitrile. Proteins were then reduced and alkylated. Dried gel pieces were digested with trypsin (Promega, Madison, WI, USA), and digests were immediately spotted onto 600 μm anchorchips (Bruker Daltonics, Bremen, Germany). MALDI-TOF/TOF-MS was carried out on a time-of-flight Ultraflex II mass spectrometer (Bruker Daltonics, Bremen, Germany). The acquired mass spectrum was processed using the software FlexAnalysis v. 2.4 (Bruker Daltonics, Bremen, Germany). The resulting peptide mass lists were submitted to a GPS explorer workstation equipped with the MASCOT search engine (Matrix Science, London, UK) to search the National Center for Biotechnology Information non-redundant protein database (NCBInr) (17 294 654

Malate and citrate in cells were extracted according to the method of Anoop et al. [22]. Briefly, the frozen cells were ground with liquid nitrogen, and homogenized with 80% (v/v) ethanol. After the homogenate was centrifuged at 12000 ×g for 5 min at 4 °C, some of the supernatant was removed to determine the protein concentration. The remaining supernatant was vortexed and boiled at 80 °C for 15 min. After samples were centrifuged at 12000 ×g for 5 min, the supernatant was passed through a 0.45 μm filter, and the filtrate was used to measure the malate and citrate contents, as described by Delhaize et al. [23]. 2.7. Statistical analysis Three independent biological replicates were carried out and 2-DE gels were run in triplicate. Values in figures are expressed as means ± SD. The statistical significance of the data was analyzed using a univariate analysis of variance (p b 0.05) (one-way ANOVA; SPSS for Windows, version 16.0, Chicago, IL, USA). 3. Results and discussion 3.1. Al-responsive proteins identified by MALDI-TOF/TOF-MS Although R. taiwanensis RS1 can tolerate high Al concentration up to 200 mM, the presence of 100 mM Al significantly inhibited its growth [8]. After 100 mM Al treatment for 24 h, the OD600 of RS1 was reduced by 75% [8]; therefore, in this study, to extend the previous study, 100 mM Al was used to examine the changes of proteins in RS1 cells. Approximately 850 proteins were resolved reproducibly on the pH 4–7 gels (Fig. 1A). Proteins present in all biological replicates and with abundance change of at least two-fold were selected as Al-responsive proteins. Thirty-three differentially regulated proteins were identified by MALDI-TOF/TOF-MS (Fig. 1A and Table 1). Close-up views of several protein spots were shown in Fig. 1B. Among them, 29 proteins decreased in abundance and four proteins increased in abundance upon Al treatment (Table 1). The identified proteins were classified into 10 groups based on their biological functions, according to the functional classification

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Fig. 1. 2-DE analysis of proteins extracted from R. taiwanensis RS1. (A) Representative 2-DE gels of RS1 in which 33 differentially expressed Al-responsive proteins showing at least 2-fold changes (p b 0.05) between the 0 and 100 mM Al treatments are marked. (B) Close-up view of selected differentially expressed proteins.

in Saccharomyces Genome Database (http://mips.helmholtz-muenchen. de/genre/proj/yeast/). As shown in Fig. 2, most Al-responsive proteins are associated with metabolism, transcription, protein fate, and biogenesis of cellular components; 18.2% of them were grouped as unclassified proteins. 3.2. Effect of Al toxicity on transcription and translation processes In response to environmental stresses, a common strategy adopted by cells is to increase the expression of stress-defense genes and to decrease protein synthesis and growth-related gene activity [24]. In the present study, the results showed that the dynamic changes of the proteome in response to high Al conditions are directly related to the process of transcription and translation in yeast RS1 (Table 1 and Fig. 2), one of the important Al toxicity markers [25,26]. For example, upon 100 mM Al exposure, proteins showing decreased relative abundance are related to mRNA modification processes (spots 4, 5 and 16), transfer RNA (tRNA) (spot 13) and protein translation (spot 7). Functioning mainly as a molecular chaperone, heat shock protein 70 (spot 7) is involved in protein modification through binding newly translated proteins to assist their proper folding and prevent aggregation/misfolding [27]. In addition, under Al toxicity, the abundance of an essential non-ATPase regulatory subunit of the 26S proteasome decreased (spot 8). This protein is part of an important degradation pathway for short-lived regulatory or damaged proteins, via the degradation of ubiquitin-conjugated proteins [28]. Furthermore, the abundance of a ubiquitin-specific protease (spot 21), which can cleave ubiquitin from ubiquitinated proteins to recycle ubiquitin in the ubiquitin–proteasome degradation process [29], also decreased under Al stress. These results suggest that the presence of high Al concentrations directly affects gene activity, protein syntheses and turnover, resulting in the inhibited-growth observed for yeast RS1. 3.3. Al is toxic to DNA defense and Golgi function DNA damage arising from exogenous or endogenous sources can block the progression of DNA replication. Some studies on the effect of Al toxicity on microorganisms have reported that DNA damage is one mechanism of Al-mediated cellular injury [30,31]. Appropriate repair of damaged DNA is therefore crucial for genome integrity. In the present study, DNA N-glycosylase and apurinic/apyrimidinic (AP) lyase (spot

23), which were involved in base excision repair by removing oxidized pyrimidines from DNA [32], showed a decreased abundance in RS1 cells under Al stress (Table 1). In addition to damage to the nucleus DNA, Al exposure also results in toxicity to the mitochondrial DNA (mtDNA). For example, the abundances of a mitochondrial ribosomal protein (spot 14) and a high mobility group (HMG) protein (spot 29) were decreased under 100 mM Al. HMG is required for the maintenance of mtDNA in growing cells and for efficient recombination of mtDNA markers in crosses [33]. These results suggest that decreases in these DNA defense proteins may lead to the breakdown of genome integrity, resulting in the inhibited-growth in RS1 under Al toxicity. The functions of these DNA-maintenance proteins in Al stress merit future study by overexpressing the related proteins. The Golgi apparatus is an important intracellular organelle, and is integral for protein modification, processing, and vesicle sorting and secretion. Al toxicity inhibits the function of the Golgi apparatus in higher plants [34]. In RS1 cells Al exposure results in decreased amounts of certain proteins related to Golgi function (spots 9 and 11). For example, the AP-3 complex subunit (spot 9) transports alkaline phosphatase to the vacuole/lysosome from the Golgi [35]. The AP-1 complex subunit (spot 11) is an important component of the complexes of the trans-Golgi network, and is involved in clathrin-dependent Golgi protein sorting [36]. These results suggest that the Golgi apparatus is one of the targets of Al toxicity in RS1 cells, and damage to the Golgi apparatus is probably one explanation for cell growth retardation. How Al toxicity leads to the Golgi apparatus damage requires further study.

3.4. Energy and glucose metabolism are affected by Al Glycogen is a major polysaccharide storage form of glucose whose storage can improve yeast survival under nutritional and physical stress conditions [37]. The Ser/Thr phosphoprotein phosphatase (spot 33) plays an important role in glycogen metabolism by regulating the phosphorylation state of glycogen synthase. Yeast cells lacking this protein have reduced glycogen levels [38]. In this study, Al treatment resulted in a decreased abundance of Ser/Thr phosphoprotein phosphatase. Furthermore, the abundance of spot 27, which is involved in the metabolism of glycogen [39], was also shown to decrease in RS1 cells in response to Al stress. These results suggest that Al stress may reduce

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Table 1 Identification of Al-responsive proteins in R. taiwanensis RS1 under 100 mM Al treatment by MALDI-TOF/TOF-MS analysis. Spot no.a

NCBI accession no. b

Protein name

c

Abundance decreased after Al exposure 1 gi|323310262 Saw1p 4 gi|37362642 Fir1p

Protein description

Fold change

17

gi|323309145

18 19

gi|323309399 gi|323309145

21 22

gi|323347679 gi|116006500

Hypothetical protein FOSTERSO_1597 Icl1p Hypothetical protein FOSTERSO_1597 Ubp11p Dcs2p

23 24 25 26 27 28

gi|6319304 gi|323348500 gi|349578322 gi|323337715 gi|6322139 gi|323331414

Ntg1p Say1p K7_Ygr111wp Ygr111wp Pcl7p YOR356wp

29 30 31 32 33

gi|6323717 gi|259148466 gi|151944974 gi|259144732 gi|259149664

Abf2p Pml39p Fbp26p Flo1p Gac1p

Ubiquitin C-terminal hydrolase Non-essential protein, regulated by Msn2p, Msn4p, and the Ras-cAMP-cAPK signaling pathway DNA repair protein Protein of unknown function Protein of unknown function Protein of unknown function Cyclin like protein interacting with Pho85p Putative mitochondrial electron transfer flavoprotein-ubiquinone oxidoreductase High mobility group protein Nuclear pore-associated protein Fructose-2,6-bisphosphatase Cell wall protein involved in flocculation Ser/Thr phosphoprotein phosphatase

Abundance 2 3 6 20

increased after Al exposure gi|323336966 Acf4p gi|323353698 Tal1p gi|116006499 Mdh2p gi|259145525 Nse3p

Protein involved in actin cytoskeleton organization Transaldolase Malate dehydrogenase Protein required for cell viability

MOWSE score g

M

h

Ci

111.76/4.97 91.94/5.66

94 151

10 24

48 30

−3.61 −3.60 −6.42 −3.43 −2.20 −2.23 −2.18 −2.01 −3.20 −4.09 −2.27

108.77/5.63 77.65/5.12 49.06/8.86 92.23/5.57 29.94/9.58 54.06/8.63 42.60/5.58 64.62/8.36 72.12/9.38 45.36/8.40 99.70/4.76

85.47/5.38 60.89/4.96 66.64/5.61 67.08/5.81 62.20/5.82 65.09/6.39 55.60/5.33 51.49/5.34 48.90/5.09 42.52/4.52 43.76/5.21

150 165 68 99 166 113 297 182 61 211 64

29 30 23 25 21 21 28 29 13 21 15

31 47 59 25 62 38 80 52 20 47 19

22.46/11.17

43.99/6.13

78

10

42

−2.04 −2.92

60.57/6.01 22.46/11.17

45.01/6.26 44.50/6.32

60 62

10 9

20 39

−3.36 Absence

65.23/8.50 40.97/6.15

34.36/4.96 37.94/5.47

61 62

14 11

29 38

Absence −2.31 −2.53 Absence Absence Absence

45.83/8.09 30.37/7.04 47.08/6.04 42.35/8.56 32.30/5.03 66.84/6.85

37.59/5.69 37.56/5.82 37.43/6.28 35.17/6.23 25.02/4.81 26.92/5.17

144 153 154 138 221 180

24 19 18 20 19 25

66 71 41 36 72 49

−2.77 −2.01 −2.08 −2.45 Absence

21.55/9.68 39.85/9.10 52.85/6.65 27.42/4.84 80.49/6.14

27.44/5.28 25.80/5.67 27.74/5.91 28.61/6.21 14.53/4.83

167 155 211 94 62

22 23 26 11 12

73 57 60 42 19

+2.34 Presence +2.44 +2.02

35.56/8.52 30.50/8.60 40.99/6.41 33.98/5.23

105.70/5.24 105.90/5.60 63.15/4.92 43.24/6.56

229 67 175 66

27 13 20 10

88 38 59 37

Isocitrate lyase Protein of unknown function

Dcp2p Sse1p Rpn3p Apl6p YOR142W–B-like protein Apm1p Yhr112cp Trm2p Rsm22p Yjr149wp Sto1p

Observed Mr/pI f

30.47/9.50 99.38/7.21

−2.58

gi|125863560 gi|533365 gi|323355426 gi|6321700 gi|323306777 gi|259149898 gi|323308743 gi|9755890 gi|323347722 gi|256272183 gi|323307753

Theoretical Mr/pI e

−2.52 −4.38

Protein of unknown function localized to nucleus Interacts with the poly(A) polymerase in the two hybrid system Suppressor protein of a yeast pet mutant Heat shock protein of HSP70 family 26S proteasome regulatory subunit AP-3 complex subunit TY1B protein AP-1 complex subunit Putative cystathionine beta-lyase tRNA(M-5-U54)-methyltransferase Mitochondrial ribosomal protein, small subunit Putative 2-nitropropane dioxygenase Large subunit of the nuclear cap-binding protein complex CBC Protein of unknown function

5 7 8 9 10 11 12 13 14 15 16

d

a

Spot no. is the differentially expressed protein spot number in Fig. 1A. Database accession numbers in the database NCBInr. Name of identified proteins. d Mean fold change in relative abundance is the ratio of abundance of proteins (as estimated by PDQuest analysis) between Al treatment and control from three biological repeats. “+” and “−” indicate upregulated and downregulated proteins, respectively. Absence: the spots appeared in the 2D gels of the control, but not of the Al treatment; presence: the spots appeared in the 2D gels of the Al treatment, but not of the control. e Theoretical mass (kDa) and pI of identified proteins. f Experimental mass (kDa) and pI of identified proteins. g Mascot search score against the database NCBInr. h The number of mass values matched. i The percentage of sequence coverage (%). b c

glycogen accumulation in RS1 cells, which is similar to the result of S. cerevisiae exposed to Al toxicity [37]. In the process of glucose metabolism, fructose-2,6-bisphosphatase (spot 31) catalyzes fructose 2,6-bisphosphate (Fru-2,6-P2) to fructose 6-phosphate (Fru-6-P) and Pi [40]. Fru-2,6-P2 is a signal molecule that controls glycolysis [41]. The abundance of a protein related to fructose-2,6-bisphosphatase was decreased in RS1 cells in response to Al, indicating that Al stress might inhibit glycolysis by downregulating the level of Fru-2,6-P2. In addition to glycolysis, the pentose phosphate pathway is another important glucose catabolic pathway. Jacoby et al. [42] reported that glucose is metabolized via the pentose phosphate pathway in yeast when glycolysis is blocked. In accordance with this observation, the abundance of transaldolase (spot 3), which regulates the generation of ribose and nicotinamide adenine dinucleotide phosphate (NADPH) in the pentose phosphate pathway, was increased in RS1 cells

exposed to Al stress. Increased abundance of transaldolase induced by Al was also found in rice [16]. These observations suggest that under the suppression of glycolysis by high Al concentrations, RS1 cells might adopt the glucose catabolism through the pentose phosphate pathway, aiming to reduce the energy-dependence. Alternatively, NADPH is required for RS1 cells to remove reactive oxygen species produced under Al toxicity. The glyoxylate cycle is also an important energy metabolic pathway, and uses some enzymes associated with the tricarboxylic acid (TCA) cycle. Isocitrate lyase (spot 18), the first key enzyme of the glyoxylate cycle, converts isocitrate into glyoxylate and succinate [43]. The overexpression of isocitrate lyase plays an important role in the survival of Pseudomonas fluorescens under Al stress [44]. In contrast, high Al concentrations led to decreased levels of isocitrate lyase, suggesting that the glyoxylate cycle in RS1 cells may be inhibited by Al stress, and the

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completion of the genome sequence, an iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography– tandem mass spectrometry approach would provide much more information about the proteome of RS1 under Al stress. 3.6. Al-stimulated citrate accumulation is an internal mechanism for tolerating Al In plants, the Al-stimulated increase in organic acids complexed with Al ions is the best documented and characterized Al-tolerant mechanism, either internally or externally [49,50]. Our previous study found that RS1 cells exposed to Al stress did not secrete organic acids to decrease the toxic form of Al ions dissolved in the medium [8]. However, the present results showed that the levels of malate dehydrogenase (MDH, spot 6) were significantly increased under Al stress in RS1 cells (Fig. 1). MDH catalyzes the NAD/NADH-dependent interconversion of malate and oxaloacetate, which is linked to the oxidation/reduction of this enzyme [51]. Tesfaye et al. [52] reported that overexpression of MDH alone could increase tolerance to Al toxicity in Alfalfa plants by enhancing organic acid synthesis, including citrate, oxalate, and malate. Citrate synthase (CS) has also been reported to play an important role in Al tolerance [22]. In this study, however, we did not detect CS as an Al-responsive protein under our criteria. Similarly, CS was not observed as an Al-responsive protein in Al-tolerant soybean roots using a proteomics approach. Al treatment did lead to the increase in the relative abundances of MDH, malate oxidoreductase and pyruvate dehydrogenase, which probably contributed to the accumulation of citrate [15]. Fig. 2. Functional classification of Al-responsive proteins in RS1 cells. Each identified protein listed in Table 1 was functionally classified according to their putative functions in the Saccharomyces Genome Database (http://mips.helmholtz-muenchen.de/ genre/proj/yeast/).

effective role of isocitrate lyase in the detoxification of Al in P. fluorescens may not be significant for R. taiwanensis RS1. The result also implies that different species may adopt distinct strategies in response to Al stress. 3.5. Response of cell wall proteins to Al stress could decrease the biosorption of Al Yeast flocculation leads to the formation of flocculent or granular cell clusters between yeast cells, which are crucial for many essential biological processes. Yeast flocculation is considered to result from an interaction between a lectin-like protein and a mannose chain present on the cell surface [45]. Flocculation protein (spot 32) at the cell surface plays an important role in the regulation of the yeast flocculation process [46]. Some studies have reported that flocculent yeast can effectively remove metal ions, such as Cu2+, Ni2+ and Zn2+, mainly because the cell wall of flocculent cells provides more adsorption sites for metal ions [46]. In the present study, under the treatment by 100 mM Al, the relative abundance of flocculation protein in RS1 cells was significantly decreased, which could result in reduced flocculation and reduced adsorption of Al ions at the cell surface, thereby decreasing the chance of Al entering the cell lumen. The importance of yeast flocculation in Al detoxification merits further study. As the first protective barrier, the cell wall plays an important role in an organism's tolerance of Al, and is also a primary target for Al toxicity. Studies have suggested Al toxicity significantly influences the expression of a number of cell wall-related proteins in plants [16,47]. Our previous study showed that the cell wall in RS1 cells was thickened in response to the high Al concentrations, probably related to high Al tolerance of RS1 [8]. Unfortunately, in the present study no proteins related to cell wall synthesis appeared to be influenced by Al stress. One explanation is that the pHs of most of these proteins lie beyond the pH 4–7 range detected in this study. Alternatively, because of the technical limitation of the gel-based 2-DE approach, proteins present at low concentrations are not easily detected in the gels [48]. With the

Fig. 3. Changes in malate dehydrogenase activity (A) and intracellular malate and citrate content (B) in RS1 cells exposed to 0 or 100 mM Al. Data shown are means ± SD of three individual measurements. Different letters above the columns indicate statistically significant differences at p b 0.05.

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Except for MDH, transcriptomic analysis of Arabidopsis roots revealed that no TCA cycle-related genes were Al responsive under Al stress [53]. However, the synthesis of citrate in P. fluorescens exposed to Al toxicity was related to increased activity and expression of many enzymes, including MDH, CS and the malic enzyme [54]. It is tempting to speculate that TCA cycle enzyme(s) and organic acids are specifically regulated by Al. In RS1 cells, MDH, rather than CS, could play an important role in Al tolerance through the accumulation of organic acids. To test the role of MDH in Al tolerance in RS1 cells, we measured its activity. As shown in Fig. 3A, the reductive activity of MDH was significantly higher than its oxidative activity either with or without Al, and the activities of both MDH forms were enhanced under Al stress, indicating that the interconversion and turnover between malate and oxaloacetate was improved in yeast RS1. The increase in the activity of MDH correlated with the observed increase in protein level revealed by proteomic analysis in RS1 cells (Table 1). At present, however, it cannot be ruled out that Al itself can induce the MDH activity [55]. MDH can convert oxaloacetate into malate and citrate; therefore, we tested whether the levels of intracellular malate and citrate were increased in RS1 cells with increased MDH activity. As shown in Fig. 3B, the citrate content was significantly higher than the malate content under control conditions, and Al stress led to an increased citrate content but not to increased malate, suggesting that the increase in MDH activity in RS1 cells exposed to high Al concentration provides more substrate oxaloacetate for citrate synthesis. Although both reductive and oxidative MDH activity increased under Al stress (Fig. 3A), MDH is mainly involved in citrate synthesis, which is possibly caused by the specific environmental factor (Al stress) influencing MDH activity in RS1 cells. For example, the accumulation of certain organic acids in S. cerevisiae was significantly influenced by growth conditions, such as nitrogen limitation, high concentrations of carbohydrate, and a relatively high pH [21]. Furthermore, Pines et al. [21] observed that the overexpression of MDH increased intracellular citrate levels in S. cerevisiae.

It is generally believed that forming an Al–citrate complex is easier than forming an Al–malate complex, and citrate is the most frequently employed organic acid for relieving Al toxicity among organic acids. Therefore, the formation of Al–citrate complexes within cells is considered as an internal Al-tolerance mechanism in certain highly Al-tolerant plants [50] and in yeast [22]. Combining the previous and present results, we suggest that RS1 cells have evolved a range of regulatory mechanisms to adapt to Al toxicity. One strategy employed by RS1 cells is to block entry of Al into the cell interior by reducing the capacity of the cell surface to adsorb Al ions and increasing the thickness of the cell wall [8]. Another strategy is to chelate Al ion by forming stable, non-phytotoxic complexes with internal citrate, where increased amounts of citrate are related to increased MDH amount and activity.

4. Conclusions Using a proteomic strategy, Al toxicity-induced changes in the total soluble protein profile were analyzed in an Al-tolerant yeast, R. taiwanensis RS1. Thirty-three proteins were identified as Alresponsive proteins under 100 mM Al. These Al-responsive proteins could be classified into 10 groups based on cellular functions involved in various biological processes, including transcription and translation, DNA defense, Golgi function, as well as glucose metabolism. In particular, MDH, one of the Al-responsive proteins, was induced under 100 mM Al, and correlated well with the increase of its activity and intracellular citrate content, suggesting that MDH could be used as an important biomarker for Al tolerance. Al-stimulated citrate accumulation could be an important internal mechanism for tolerating Al. Based on the proteomic data, we constructed a schematic model of RS1 cells' response to high Al concentrations (Fig. 4). Taken together, the results provide valuable information for further exploration of Al toxicity and tolerance in R. taiwanensis RS1.

Fig. 4. Schematic model of RS1 cells in response to high Al concentration. The abbreviations shown in the figure are the same as those in Table 1. ↑ and ↓indicate increased and decreased proteins, respectively.

C. Wang et al. / Biochimica et Biophysica Acta 1834 (2013) 1969–1975

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