responsive proteins in Arabidopsis thaliana - Springer Link

Plant Cell Rep (2009) 28:1603–1614 DOI 10.1007/s00299-009-0759-2

ORIGINAL PAPER

Proteomic identification of toxic volatile organic compoundresponsive proteins in Arabidopsis thaliana Min-Ah Park Æ Jae-Hyun Seo Æ Jong-Sug Park Æ Mi Kwon

Received: 20 May 2009 / Revised: 23 July 2009 / Accepted: 29 July 2009 / Published online: 21 August 2009 Ó Springer-Verlag 2009

Abstract The proteins that are responsive to toxic volatile organic compounds (VOC) such as formaldehyde and toluene were analyzed with proteome analysis using twodimensional difference image gel electrophoresis (DIGE) technology. Twenty-one days after germination (DAG) seedlings of Arabidopsis thaliana were exposed either to the gaseous formaldehyde or toluene in an airtight box installed in a plant growth chamber maintained at 24°C under the long day condition with relatively low light condition. Comparative expression analysis revealed 14 and 22 protein spots as proteins displaying at least 1.5-fold differences in expression upon formaldehyde and toluene treatment, respectively, compared to those of untreated control. Most of the isolated spots were successfully identified by peptide analysis using LC-MS-MS. The VOCresponsive proteins contain ATP synthase CF1, ribulose1,5-bisphosphate carboxylase/oxygenase, photosystem II light harvesting complex, and enolase, which are components of photosynthesis and carbohydrate metabolism. Despite the relatively low light intensity was applied, many identified VOC-induced proteins were previously known to

Communicated by J. R. Liu. J.-H. Seo M. Kwon (&) Department of Forest Products, College of Forest Science, Kookmin University, Seoul 136-702, Korea e-mail: [email protected] M.-A. Park Department of Agricultural Sciences, College of Agriculture, Korea National Open University, Seoul 110-791, Korea J.-S. Park Functional Biomaterial Division, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea

be up-regulated upon high light stimulus. In addition, proteins involved in the toxin catabolic process and stress hormone-related proteins were identified as tolueneinduced proteins. Although the exact function of most of the VOC-responsive proteins identified in these experiments had not been characterized, the protein expression analysis using DIGE was clearly demonstrated that plants are capable of responding actively to VOCs at translational level, and identified proteins may provide valuable tools to account for the effects of abiotic stress caused by air pollutants such as VOCs in plant. Keywords Arabidopsis thaliana 2D-DIGE Formaldehyde Proteome Toluene Volatile organic compound

Introduction Volatile organic compounds (VOCs), a group of chemical compounds that are easily evaporated in the air due to their high vapor pressures, cause harmful effects to the environment and living organisms. VOCs originate from both outdoor sources, mainly from fuel emissions, and indoor sources such as furnishings, machines, solvents cleaning agents, and clothes (Wood et al. 2002). The outdoor air contamination from these chemical mixtures can cause photochemical smog when they react with nitrogen oxidation materials to give oxidation products such as ozone and peroxyacetyl nitrate, as well as global warming (Gray et al. 2005). In addition, the long-term exposure to these chemical mixtures has been recognized as a component of indoor air pollution leading to sick-building syndrome with symptoms of headaches, irritation of the mucosa, and respiratory problems (Wolkoff 1995; Weschler and Shields

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1996; Wippermann et al. 1999), even at very low concentrations in poorly ventilated indoor areas. Formaldehyde, a representative VOC that causes both indoor and outdoor air pollution, is a suspected carcinogen (Wippermann et al. 1999) since it causes mutation, alkylation, and cross-links that destroy the functions of membranes, proteins, and nucleic acids when it reacts with amino and sulfhydryl groups of biological molecules (Heck et al. 1990; Hickman et al. 2004). The long-term exposure of gaseous formaldehyde has been reported to promote allergic inflammatory effects and enhance pre-existing allergic symptoms in the currently establishing levels in a murine model (Gu et al. 2008). Therefore, it is regarded not only as a pollutant but also as a toxic compound to living organisms. Unlike other VOCs, formaldehyde can be produced by endogenous metabolic processes in plant, including the oxidative demethylation of amino acids, oxidation of methanol that derived mainly from pectin demethylation, oxidation of one-carbon compounds (Fall and Benson 1996; Sa´rdi and Tyiha´k 1994), glyoxylate decarboxylation (Prather and Sisler 1972), and cytochrome P450-dependent oxidation of herbicides (Clejan and Cederbaum 1993). In general, exposure to excess amounts of formaldehyde has been reported to cause severe physiological changes that are detrimental to plants to cause, for example, forest decline (Cape 2003). However, some of the foliage plants are reported to effectively remove air-borne VOCs, even indoors where the light intensity is not enough for active photosynthetic processes. Recent biochemical studies imply that the plant detoxification of exogenous formaldehyde may be achieved at least partially by activation of C1 metabolism in vivo to utilize carbon in the formaldehyde molecules as a carbon source (Achkor et al. 2003). For example, a feeding experiment of common spider plants (Chlorophytum comosum) with 14C-formaldehyde demonstrated that formaldehyde can be incorporated into the metabolism of photosynthetic cells and used as a carbon source as it afforded 14C-labeled product derived from C1 metabolism (Giese et al. 1994). However, it remains to be elucidated how indoor plants can actively remove the airborne VOCs in the condition where the photosynthetic power is very limited compared to the outdoor environmental condition. The purpose of this study was to isolate the proteins responsive to exogenously treated gaseous VOCs in Arabidopsis thaliana and to gain a better understanding of the plant responses to abiotic stress caused by air pollutants such as VOCs. Formaldehyde was used as a representative toxic VOC as they commonly occur in both the normal household and outdoor environment (Cape 2003; Kwon et al. 2008). For the comparison, toluene was also used to treat A. thaliana as it is also frequently detected toxic VOC in both outdoor and indoor. To identify the VOC-responsive proteins in A. thaliana, two-dimensional difference

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image gel electrophoresis (2D-DIGE) technology was utilized because of its sensitivity and reliability based on the technique of analyzing up to two pools of protein samples simultaneously on a single 2D-gel to minimize the problems of gel-to-gel variability (Amme et al. 2006). The results of this study strongly imply that the plant can actively respond to toxic VOCs by altering the expression level of many proteins and enzymes involved in photosynthesis and carbon metabolism, as well as toxin catabolic process and stress hormone-related proteins.

Materials and methods Plant growth and exposure to volatile organic compounds Seeds of wild type (Col) A. thaliana were sown in soil (Sunshine Mix #5, SunGro, Bellevue, WA, USA) in 9-cmdiameter pots that were presoaked for 4 days at 4°C before being transferred to a plant growth chamber maintained at 24°C under a 16 h photoperiod with low light intensity (approximately 47.3 lmol m-2 s-1) compared to the standard plant growth facility. The plastic wrap was removed at 5 days after germination (DAG) and the pots were subirrigated with tap water as required. At 21 DAG, ten pots were placed in a box of dimensions 56 9 35.5 9 14 cm3 with each pot containing 4–5 seedlings of 21 DAG A. thaliana. A 3,108 ll sample of either formalin (37% formaldehyde) or toluene was equally divided into eight Eppendorf tubes to give 388.5 ll per tube. The tubes containing VOCs were then immobilized on the inside of the plastic box with the cap opened. The box was then sealed with plastic wrap so that the plants were exposed to the gaseous VOCs as they were evaporated within the box (Fig. 1). A mock control was prepared by adding distilled water instead of VOCs in the Eppendorf tubes. The sealed boxes were placed back into the plant growth chamber. After 24 h, the plastic wrap was carefully removed under the fume hood and the aerial parts of the 21 DAG seedlings of A. thaliana were harvested carefully and immediately transferred into liquid nitrogen. Samples were stored at -80°C until needed. Protein isolation and labeling The plant tissues were homogenized in the presence of liquid nitrogen and used for the extraction of total proteins using Trizol reagent (Invitrogen Life Technologies, Rockville, MD, USA) according to the manufacturer’s recommendation. Total protein concentration was determined using PlusOne 2-D Quant kit (GE Healthcare, Piscataway, NJ, USA) according to the procedure described.

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Fig. 1 VOC treatment and the phenotypes of 27 DAG Arabidopsis thaliana seedlings exposed to VOCs. The A. thaliana seedlings were exposed to gaseous VOCs in an airtight box for 24 h in a growth chamber with low light intensity (a) and the evaporated (red) and residual (blue) amounts are shown after 24-h treatment (b). Gross phenotypes of 27 DAG Arabidopsis seedlings after VOC treatment (c) and their fluorescence microscopic images after staining with FDA (d). Although the toluene concentration in the airtight box was much higher than that of formaldehyde, the phenotypic changes, based on the color of leaves and the degree of viability, were more severe in the formaldehyde-exposed seedlings. Vertical bars in b indicate standard deviation of three replications. Scale bars indicate 200 lm

Protein was dissolved in standard cell lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 30 mM Tris/HCl. After adjusting the pH of protein lysate between 8.0 and 9.0, the protein was labeled using Cy3, Cy5, and Cy2 in the dark. Briefly, 400 pmol CyDIGE flours of Cy3 or Cy5 were added to 50 lg of protein lysate extracted from either the control or VOC-treated seedlings, respectively. For internal standard, 25 lg of each protein of control and VOC-treated samples were mixed together and labeled with 400 pmol of Cy2 in CyDIGE flour working solution. The internal control labeled with Cy2 and the protein samples labeled either with Cy3 or Cy5 were incubated for 30 min at 4°C in CyDIGE flour working solution. After stopping the labeling reaction by adding 1 ll of 10 mM lysine to the mixture, the samples were incubated for 10 min at 4°C in the dark. Three protein samples labeled differentially (Cy3, Cy5, and Cy2) were combined together to make 150 lg of labeled protein mixtures. After adding the same amount of 29 sample buffer containing 2 M thiourea, 7 M urea, 2% (v/v) pH 3– 10 pharmalytes, 2% (w/v) DTT, and 4% (w/v) CHAPS to the labeled protein mixture, it was incubated for 10 min on ice. After readjusting the volume of the labeled protein to

450 ll by adding rehydration buffer containing 8 M urea, 4% CHAPS, 1% pH 3–10 pharmalytes, and 0.002% bromophenol blue, the protein samples were immediately used for isoelectric focusing. Protein labeling was repeated three times with dye swapping, as listed in Table 1. 2D-gel electrophoresis and imaging One hundred fifty micrograms of rehydrated proteins, as determined with a PlusOne 2D-Quant kit (GE Healthcare, Piscataway, NJ, USA), was loaded onto a 24-cm nonlinear pH 3–7 Immobiline Drystrip (GE Healthcare, Piscataway, NJ, USA). The strip was rehydrated in the presence of sample for 16 h at room temperature. The first dimension was focused on an Ettan IPGphor system (GE Healthcare, Piscataway, NJ, USA) for a total of 88 kVh at 20°C with a linear increase of voltage from 0 to 300, 600, 1,000 and 8,000 V for 24 h. The IPG strip was then equilibrated for 10 min in a buffer containing 50 mM Tris/HCl (pH 8.8), 6 M urea, 20% glycerol (w/v), 2% SDS (w/v), and 1% DTT (w/v). The strip was then equilibrated in a second buffer containing 50 mM Tris/HCl (pH 8.8), 6 M urea, 20% glycerol (w/v), 2% SDS (w/v), and 2.5% iodoacetamide

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Table 1 Design of 2D-DIGE experiments for three technical replications Analysisa

Cy2 (standard)

Cy3 (sample)

Cy5 (sample)

1

Control (25 lg) ? formaldehdye-treated (25 lg)

Control (50 lg)

Formaldehdye-treated (50 lg)

2



Control (50 lg)

3


Control (50 lg)


4

Control (25 lg) ? toluene-treated (25 lg)

Control (50 lg)

Toluene-treated (50 lg)

5



Control (50 lg)

6


Control (50 lg)


Each sample was covalently labeled with a different one of the following fluorophores: Cy2 (a mixture of equal amounts of protein extracts from control and treatment), Cy3 (control), and Cy5 (treatment). The second set of experiments was performed with dye swapping (Cy2 with a mixture of equal amounts of protein extracts from control and treatment, Cy3 for treatment, and Cy5 for control). The third set of experiments was performed exactly the same as the first set a

For each set of analyses, three gels were performed to reduce technical variations

(w/v) for an additional 10 min. The second dimension separation was performed on 12% SDS polyacrylamide gel upon application of 8 watts per gel using an Ettan DALT six system (GE Healthcare, Piscataway, NJ, USA). Kaleidoscope Prestained Standards (GE Healthcare, Piscataway, NJ, USA) were electrophoretically separated in parallel to calibrated masses of the Arabidopsis proteins against known molecular weights. For image analysis, the gel was immediately placed on the plate of a Typhoon 9400 scanner (GE Healthcare, Piscataway, NJ, USA) and scanned three times in the fluorescence acquisition mode with the appropriate emission filter for Cy2, Cy3, and Cy5 each time. The fluorescence signals of the three differentially CyDyelabeled protein samples were imaged using a laser scanner recording band pass-filtered emission wavelengths of 520 nm (Cy2), 580 nm (Cy3), and 670 nm (Cy5), respectively (Typhoon 9400, GE Healthcare, Piscataway, NJ, USA). Scanned images were then analyzed using Decyder program V6.5 (GE Healthcare, Piscataway, NJ, USA). Proteins were poststained with silver staining and the spots of interest were excised manually and subjected to mass spectrophotometric protein identification. Protein spot analysis by MALDI-TOF and LC-MS-MS The excised protein spots were digested in-gel with trypsin (Promega Madison, WI, USA) according to the method described in Shevchenko et al. (1996). The enzymatically digested samples were then analyzed by either MALDITOF or by LC-MS-MS. All samples were analyzed using a Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Fostercity, CA, USA). Masses were measured in the reflectron/delayed extraction mode with an accelerating voltage of 20 kV, grid voltage of 76.0%, and delay time of 150 ns. A two-point external standard for calibration was used with des-Argl-Bradykinin (m/z 904.4681) and neurotensin (m/z 1296.6853). Peptides were

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selected in the mass range between 700 and 3,500 Da. For data processing, the software package Data Explore was used. Database searches were performed using Protein Prospector (http://prospector.ucsf.edu). The following search parameters were applied: NCBI was used as the protein sequence database; a mass tolerance of 50 ppm and one incomplete cleavage were allowed; and the possible modifications were acetylation of the N-terminus, alkylation of cysteine by carbamidomethylation, oxidation of methionine, and the pyroGlu formation of N-terminal Gln. For the LC-MS-MS experiments, the integrated system used consisted of an auto switching pump, nano pump, autosampler (Tempo nano LC system, Applied Biosystems, Fostercity, CA, USA), and a hybrid Quadrupole-TOF MS/ MS spectrometer (QStar Elite, Applied Biosystems, Fostercity, CA, USA) equipped with a nano-electrospray ionization source and fitted with a fused silica emitter tip (New Objective, Woburn, MA, USA). Fractions were reconstituted in solvent A (water/acetonitrile [ACN] (98:2 v/v), 0.1% Formic acid) and then injected into an LC-nano ESIMS/MS system. Solvent A consisted of water/ACN (98:2, v/v) with 0.1% formic acid for the high aqueous mobile phase. Peptides were first trapped on a Zorbax 300SB-C18 ˚ , Part trap column (300 lm i.d. 9 5 mm, 5 lm, 100 A number 5065-9913, Agilent Technologies, Atlanta, GA, USA) and washed for 10 min with 98% solvent A (water/ ACN (98:2, v/v), 0.1% formic acid) and 2% solvent B (water/ACN (2:98, v/v), 0.1% formic acid) at a flow rate of 10 ll/min, and then separated on a Zorbax 300SB-C18 ˚, capillary column (75 lm i.d 9 150 mm, 3.5 lm, 100 A part number 5065-9911, Agilent Technologies, Atlanta, GA, USA) at a flow rate of 300 nl/min. The LC gradient was run with solvent B at 2–35% for 120 min, then from 35 to 90% for 10 min, 90% for 15 min, and finally 5% for 35 min. The resulting peptides were electrosprayed through a coated silica tip (FS360-20-10-N20-C12, PicoTip emitter, New Objective, Woburn, MA, USA) at an ion

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spray voltage of 2,300 eV. Mass data were acquired automatically using Analyst QS 2.0 software (Applied Biosystems, Fostercity, CA, USA). For data acquisition, the mass spectrometer was set in the positive ion mode at a selected mass range of 400–1,600 m/z for a 1-s TOF-MS survey scan to detect precursor ions. Information-dependent acquisition of the MS/MS data was performed on the five most abundant peptides exceeding 20 counts with ?2 to ?4 charge states within a 100–2,000 m/z value window. Fragment target ions were dynamically excluded for 60 s with a 100 ppm mass tolerance. Curtain gas was set at 22, and nitrogen was used as the collision gas. Chlorophyll content The chlorophyll content was measured according to the method described previously (Hiscos and Israelstam 1979). Briefly, 100 mg of leaf tissue was placed in a glass vial containing 7 ml DMSO (Sigma, St Louis, MO, USA) and chlorophyll was extracted into the fluid by incubation at 65°C for 30 min. The liquid containing the extracted chlorophyll was transferred to a graduated tube and made up to a total volume of 10 ml with DMSO. One-milliliter sample of chlorophyll extract was transferred to a cuvette and OD values at 645 and 663 nm were read using a spectrophotometer (Agilent Technologies, Atlanta, GA, USA) with DMSO blank. The chlorophyll contents were calculated using the equation proposed by Arnon (1949). Viability test using fluorescein diacetate A vital staining technique with fluorescein diacetate (FDA) (Sigma, St Louis, MO, USA) was used to determine the viability of leaf tissues before and after VOC treatment. FDA stock solution (5 mg/ml in acetone, total volume 40 ll) was diluted with distilled water to a final concentration of 0.002% (w/v) in a microtube. The leaf was placed into the FDA solution and incubated for 30 min at room temperature in the dark. The stained leaf was then photographed under a fluorescent microscope (DM2500, Leica, Wetzlar, Germany).

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box was not circulated, the concentration of VOCs expected to be the similar for all plants as the VOC aliquot was evenly distributed within the box. After 24 h, the residual amounts of formaldehyde and toluene were 2,941 and 532.5 ll, equating to evaporated amounts of 167 and 2,575.5 ll, to give a concentration within the plastic box of approximately 7.4 9 10-5 and 0.87 mM, respectively (Fig. 1b). After 24-h exposure, the overall phenotypes of the 21 DAG A. thaliana seedlings of either formaldehyde treated (Fig. 1c) or toluene treated (Fig. 1d) were not seriously altered compared with those of the control (Fig. 1c), as judged by the phenotypes with the naked eye (Fig. 1c). Interestingly, even though the toluene concentration within the box was much higher than that of formaldehyde, the toluene-treated A. thaliana looks much healthier (Fig. 1c) than the formaldehyde-treated samples (Fig. 1c). Viability testing using FDA clearly visualized the living and intact cells with green fluorescent image in both control and toluene-treated seedlings (Fig. 1d). However, the formaldehyde-treated samples showed a significant loss in viability compared to both control and toluene-treated samples (Fig. 1d), demonstrating the more toxic effects of formaldehyde than those of toluene. In addition, total chlorophyll content of formaldehyde-treated samples was significantly decreased compared with that of control, while total chlorophyll content was not significantly altered in toluenetreated samples (Fig. 2), consistent with more severe phenotypic changes in formaldehyde-treated plants (Fig. 1c). Interestingly, the significant decrease of chlorophyll content was mainly caused by the significant reduction of chlorophyll b rather than chlorophyll a (Fig. 2). Considering the gross phenotype and viability test results together

Results Phenotypic changes after VOC treatment For gaseous VOC treatment, ten 9-cm-diameter pots containing four seedlings of 21 DAG A. thaliana were put into a plastic box (56 9 35.5 9 14 cm3) containing 3.108 ml of either 37% formaldehyde or 100% toluene and the top of the box was sealed with plastic vinyl wrap (Fig. 1a). The seedlings were incubated for 24 h within a plant growth chamber with relatively low light condition. Although the air in the

Fig. 2 Total chlorophyll amounts after VOC treatment. Total chlorophyll amount and both chlorophyll a and b were significantly decreased after 24-h exposure to VOC with the toluene-treated seedlings exhibiting steeper changes. The experiment was repeated three times with total rosette leaves of control, formaldehyde-, and toluene-treated seedlings. Measurement was repeated three times with each three duplicates. Vertical bars indicate standard deviation of three replications. Statistical analysis indicates that the results are reliable with 95% confidence intervals

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Our previous SDS-PAGE experiment clearly demonstrated that overall protein expression pattern started to be altered after 6 h exposure to formaldehyde and the changes in expression became more clear at 24 h of treatment (Kwon et al. 2007). In this study, thus, A. thaliana seedlings were used to isolate formaldehyde-responsive proteins after 24 h. Protein extracts were prepared from the aerial parts of the control and formaldehyde-treated samples and were separated by 2D-gel electrophoresis after labeling with CyDye, as described in the ‘‘Materials and methods’’. To detect proteins differentially expressed upon formaldehyde treatment, 2D-DIGE patterns from mock control and formaldehyde treatment were compared. All experiments were repeated three times with three individual set of experiments, as listed in Table 1, to reduce the technical variation and the variations caused by sample differences. A representative image from the 2D-DIGE gel of the formaldehyde treatment is shown in Fig. 3. Equal amounts (50 lg) of control (Cy3), formaldehyde-treated samples

(Cy5) and the mixture (Cy2) of the individually labeled protein samples were loaded on the gel to afford 150 lg of protein from each of control and formaldehyde treatment and were separated by 2D-gel electrophoresis (see ‘‘Materials and methods’’). Figure 3a is an overlay image of the total labeled proteins visualized for all fluorophores (Cy3, Cy5, and Cy2), and Fig. 3b–d are visualized only for individual cyanine dyes: Cy3 (green) for control, Cy5 (red) for formaldehyde treatment, and Cy2 (blue) for the standard mixture (Fig. 3). Upon formaldehyde treatment, the overall protein expression pattern was greatly altered compared to the control (Fig. 4). With the analysis of nine gels for three set of experiments, a total of 14 spots were identified as proteins displaying at least 1.5-fold different expression between control and formaldehyde treatment (Fig. 4). Among these 14 spots, 10 appeared to be induced, while 4 protein spots showed decreased accumulation by formaldehyde treatment. The comparison of the normalized spot volume of selected protein spots revealed nearly equal values from the three replications (data not shown). The selected 14 spots were digested for identification of the amino acid sequences of the selected proteins. Many of the selected proteins were identified using MALDI-TOF and LC-MS-MS (Table 2). Among the 14 selected protein spots from the formaldehyde treatment, only 12 were successfully sequenced and the other 2 with relatively less

Fig. 3 Results of 2D-DIGE analysis for formaldehyde-responsive proteins in A. thaliana. Equal amounts (50 lg) of untreated control (Cy3), formaldehyde treatment (Cy5), and a mixture (Cy2) of the individually labeled samples were loaded on the gel to give 150 lg of protein from each of the untreated control and formaldehyde treatment. Separation by 2D-gel electrophoresis was performed with

a pI range of 3–7 as the nonlinear strip. The experiments were repeated three times with dye swapping. Labeled proteins were visualized using parameters appropriate for all fluorophores (a), or the individual cyanine dyes: Cy3, green, for the untreated control (b), Cy5, red, for the formaldehyde treatment (c), and Cy2, blue, for the internal standard mixture (d)

with the amount of VOCs in the box, therefore, formaldehyde seems to be more toxic to the A. thaliana plants than toluene in the condition tested. 2D-DIGE protein patterns of formaldehyde-treated plants and spot identification

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Fig. 4 Image analysis of 2Dgels with proteins labeled with Cy3 and Cy5, for control and formaldehyde-treated samples, as well as Cy2 as the internal control. Spots were selected for the proteins displaying at least 1.5-fold differences between the control and treated samples. For each spot selected, the expression levels displayed as pixel volume were compared between the untreated control (left) and the treated sample (right) by scanning with Typoon 9400 (GE Healthcare, Piscataway, NJ, USA) and its analysis program

expression level could not be sequenced, probably because of the low abundance of the protein amount isolated from the gel. Only the peptide spots exceeding the default significant probability-based molecular weight search (MOWSE) score value (p \ 0.05) were considered primarily. Using the peptide sequence information obtained, a database search using the BLAST algorithm was performed to find homologous sequences for each identified protein spot. The identified peptide sequences, their coverage, predicted molecular weight, and pI values are listed in Table 2. Among the proteins showing increased expression, seven of them were ATP synthase (Table 2). In addition, spot #557 in Table 2 were ribulose-1,5-bisphosphate carboxylase/oxygenase, the first enzyme of the carbon fixation in the Calvin cycle operating in the chloroplast of the leaves, implying an activation of carbon fixation upon formaldehyde treatment. On the other hand, the translation elongation factor and fructose-bisphosphate aldolase were identified as proteins with significantly reduced expression upon formaldehyde treatment (Table 2). 2D-DIGE patterns of toluene-treated plants and spot identification To compare the effects of formaldehyde, toluene was chosen for this experiment since it is another representative VOC that cause air pollution (Olsgard et al. 2008). In addition, it is also easily detected in various household products from indoor environment (Kwon et al. 2008). Like formaldehyde, toluene is a toxic compound that can cause harmful diseases, including respiratory problems, vision

disorders, headaches, vomiting, and dizziness (Kwon et al. 2008; Olsgard et al. 2008). However, the effects of toluene on plants have not yet been characterized. In our previous study, the phenotype of Arabidopsis seedlings exposed to toluene was not significantly altered, even after 48-h treatment (Kwon et al. 2007), although the amount of volatiles in the air was much higher than that of formaldehyde (Fig. 1). In addition, toluene-treated seedlings appeared to be healthier than formaldehyde-treated seedlings at 24-h treatment (Fig. 1), implying a more active detoxification process upon toluene treatment. For DIGE analysis of toluene-treated samples, protein extracts were prepared from the aerial parts of the control and toluenetreated A. thaliana and were separated by 2D-gel electrophoresis, as previously described. Again, the experiment was repeated three times with three set of dye-swapping experiments. The comparison of the normalized spot volume of selected protein spots revealed nearly equal values from the three replications (data not shown). A representative image from a 2D-DIGE gel of the toluene treatment is shown in Fig. 5. The selected channels correspond to the untreated control (Cy3, Fig. 5b), toluenetreated (Cy5, Fig. 5c), and mixed (Cy2, Fig. 5d) samples. The image analysis revealed 22 spots showing at least 1.5fold different protein expression level by comparing the treated and control samples based on the results from nine gels for three set of experiments (Fig. 6) with significance. Image analysis based on the pixel volume for each spot demonstrated that 20 of these were increased but the other 2 showed decreased accumulation in the toluene-treated plants when compared with controls (Fig. 6).

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Table 2 2D-DIGE analysis and identification of selected formaldehyde responsive proteins by MALDI-TOF and LC-MS/MS Spot # T-test

Av. ratioa Identified protein

Accession number

Identified peptideb

139

0.022

-1.56

Translation elongation factor

NP_567820 ALAATGGDLEKAQEFLRSQDFAAE VAAQTAAKPKSEMPAVK

5.88

103,782

4.95

435

0.0068

2.58

55,328

5.19

0.013

3.22

NP_051044 IAQIPVSEAYLGVINALANPIDGRLIE SPAPGIISRTNKPQFQEII NP_051044 IAQIPVSEAYLGRLIESPAPGIISRVIN ALANPIDGRVGSAAQIK

11.83

441

ATP synthase CF1 alpha subunit ATP synthase CF1 alpha subunit

10.45

55,328

5.19

442

0.0062

3.23

ATP synthase CF1 alpha subunit

NP_051044 ADEISNIIRASSVAQVVTSLQEREAYP GDVFYLHSRIAQIPVSE

24.85

55,328

5.19

527

0.0078

1.76

Putative enolase

AAL59917

53.15

47,728.3

5.61

539

0.0062

1.51

AGAVVSGIPLYKHIANLAGNPKAG NVNNIIGPALIGKDGGSD ATP synthase beta chain 1, NP_568203 KGSITSVQAIYVPADDLTDPAPATTF mitochondrial AHLDATTVLSRNLQDIIA

77.16

59,670.9

6.18

541

0.015

2.2

ATP synthase CF1 beta subunit

NP_051066 ATNLEMESKAVAMSATEGLKRESG VINEQNLAESKFVQAGS

54.22

53,957

5.38

546

0.013

1.68

53,957

5.38

0.0073

1.56

NP_051066 SAPAFIELDTKSQPFFVAEVFTGSSVP VGGATLGRTNPTTSNP NP_051067 VTPQPGVPPEEAGAAVAAESSTGTW TTVWTDGLTSLDRYKG

79.32

557

ATP synthase CF1 beta subunit Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

69.52

52,955

5.88

1347

0.02

1.5

Glutathione transferase

NP_850479 AITQYLAEEYSEKGEKVLATLYEK DLQFELIPVDMRASIKVH

28.52

29,231.5

8.5

1367

0.033

1.84

Putative H?-transporting ATP synthase

AAL69493

27,408.5

9.35

1621

0.0035 -1.71

Fructose-bisphosphate aldolase, putative

NP_568049 AKANSLAQLGKASSYADELVKATPE QVAAYTLKDRATPEQ

% Cov Mr (Da)

LEAPQLAQIAKLTDTQLAEVRSATAA 24 SSYAMALADVAKSVV 32.91

42,987.86 6.79

a

Minus and positive values of the Av. ratios indicate down- and up-regulation of proteins upon formaldehyde exposure, respectively

b

Only part of the peptide sequences obtained are listed

The 22 spots showing at least 1.5-fold changes in protein expression by toluene compared to untreated control were chosen and digested for identification of the peptide sequences. However, five spots with relatively low amount of extracted proteins were not processed for the peptide sequencing. The peptide sequences of most proteins with reasonable expression levels were successfully identified using LC-MS-MS (Table 3). Only the peptide spots exceeding the default significant, probability-based MOWSE score value (p \ 0.05) were considered primarily. Using the peptide sequence information obtained, a database search using the BLAST algorithm was performed to find homologous sequences for each identified protein spot and the results are listed in Table 3. Unlike formaldehyde treatment, the toluene-induced proteins identified included more diverse enzymes. The identified peptide sequences, their coverage, predicted molecular weight, and pI values are listed in Table 3. The proteins with increased expression level upon toluene treatment includes three ATP synthase CF1 alpha subunits, ribosomal protein L12, putative ribose 5-phosphate isomerase, putative RNA binding protein, In2-1 protein, photosystem II light harvesting complex, ribulose-1,5-

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pI

bisphosphate carboxylase/oxygenase large subunit, ethylene-responsive transcriptional factor, glutathione transferase, F-box family protein, and putative H?-transporting ATP synthase (Table 3). Thioglucoside glucohydrolase and calcium ion binding protein with two unidentified spots displayed significantly reduced expression upon toluene treatment.

Discussion Although the harmful effects of VOCs on plants are well recognized, information on their physiological and biochemical effects remains very limited at the cellular and molecular levels. As a first step to understand the plant response to gaseous toxic VOCs, proteins that are responsive to toxic formaldehyde and toluene were identified from 21 DAG seedlings of A. thaliana using 2D-DIGE technology. The 2D-DIGE technology enables the analysis of two pools of protein sample simultaneously on a single gel, thereby minimizing the problem of gel-to-gel viability (Amme et al. 2006). In addition, the lower detection limit of 2D-DIGE and the normalization of spot intensities facilitate

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Fig. 5 Results of 2D-DIGE analysis for toluene-responsive proteins in A. thaliana. Equal amounts (50 lg) of untreated control (Cy3), toluene treatment (Cy5), and a mixture (Cy2) of the individually labeled samples were loaded on the gel to give 150 lg of protein from each of the untreated control and toluene treatment. Separation by 2D-gel electrophoresis was performed with a pI range of 3–7 as the

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nonlinear strip. The experiments were repeated three times with dye swapping. Labeled proteins were visualized using parameters appropriate for all fluorophores (a), or the individual cyanine dyes: Cy3, green, for the untreated control (b), Cy5, red, for the toluene treatment (c), and Cy2, blue, for the internal standard mixture (d)

Fig. 6 Image analysis of 2Dgels with proteins labeled with Cy3 and Cy5, for control and toluene-treated samples, as well as Cy2 as the internal control. Spots were selected for the proteins displaying at least twofold differences between the control and treated samples. For each spot selected, the expression levels displayed as pixel volume were compared between the untreated control (left) and the treated sample (right) by scanning with Typoon 9400 (GE Healthcare, Piscataway, NJ, USA) and computer calculations with its analysis program

the generation of more reliable data than those are obtained with the silver staining method (Lilley and Dupree 2006). Although the proteomic approach is not sufficient to identify all of the relevant changes in protein abundance and the responses of plant to VOCs, the identified proteins in this experiment provide meaningful information for the

understanding of overall changes in protein expression. Among the differentially abundant proteins, ATP synthase CF1 alpha subunit is consistently identified as up-regulated protein upon formaldehyde and toluene treatment. The level of the ATP synthase CF1 alpha subunit was previously shown to be increased with the increased light

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1612

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Table 3 2D-DIGE analysis and identification of selected toluene responsive proteins by MALDI-TOF and LC-MS/MS Accession number

Identified peptideb

% Cov Mr (Da) (score)

pI

-1.54 Thioglucoside glucohydrolase

CAH40824

FEKGFIFGVASSAYQVEGGRGRGLNVWD SFTHRFPEKFSIAW

63

53,521

5.91

-1.5

NP_001031199 APMIDNPEFKDDPELYVFPKLLSDD VDQTKSGSLFDNVLVSD

13.92

49,142.57 5.58

4.12 ATP synthase CF1 alpha subunit

NP_051044

IAQIPVSEAYLGRLIESPAPGIISRVINALAN PIDGRVGSAAQIK

10.45

55,328

5.19

0.009


NP_051044

IAQIPVSEAYLGRVINALANPIDGRLIESPA PGIISRTNKPQFQE

11.83

55,328

5.19

409

0.0064


NP_051044

ADEISNIIRASSVAQVVTSLQEREAYPGD VFYLHSRIAQIPVSE

24.85

55,328

5.19

952

0.033

1.98 Ribosomal protein L12

CAA48183

AVEAPEKIEKIGSEISSLTLEEARVLSD VGDIPVQEIRRLLQVGI

12.83

19,698.54 5.51

1259 0.009

1.81 Putative ribose 5phosphate isomerase

AAM65920

IDLAIDGADEVDPNLDLVKGRGGA LLRMVEAVADKFIVVAD

26.81

29,319.80 5.73

1265 0.0064

1.94 Putative RNA binding protein; chloroplast

AAM66970

SKGFGFVTYDSSQEVQNAIKSSFGSSGSGYG 30.45 GGGGSGAGSGN

1268 0.021

1.69 In2-1 protein

AAM61679

1270 0.0047

2

1327 0.027

Spot #

T-test

Av. ratioa

256

0.009

385

0.009

397

0.0064

401

Identified protein

Calcium ion binding; CRT1

5.06

8.51

27,091.05 4.99

GPSGSPWYGSDRGLATDPEAFAEL KFGEAVWFKELEVIHSR

21.51

28,053.91 5.28

1.61 Ribulose-1,5-bisphosphate NP_051067 carboxylase/oxygenase large subunit

AGVKEYKLTYYTPEYETKDTDILAAFRLT YYTPEYETKDTDI

22.96

52,955.06 5.88

1331 0.009

1.71 Ethylene-responsive transcription factor

AAF79440

AAVGAPDHLGDCPFSQRTLFSLDSFEKVS AVDLSLAPKSNIFG

20.68

50,160.29 6.81

1347 0.0064

1.96 Glutathione transferase

NP_850479

AITQYLAEEYSEKGEKVLATLYEKDLQF ELIPVDMRVLDVYE

28.52

29,231.58 8.5

1410 0.024

2.3

NP_198978

2.64

52,305.88 4.96

1417 0.009

2.51 Ribulose-1,5-bisphosphate NP_051067 carboxylase/oxygenase large subunit

VTPQPGVPPEEAGAAVAAESSTGTWTT VWTDGLTSLDRLTY

36.95

52,955.06 5.88

1441 0.036

2.21 Putative H?-transporting ATP synthase

AAL69493

LEAPQLAQIAKLTDTQLAEVRSATAA SSYAMALADVAKSVV

24

27,408.52 9.35

1456 0.044

1.54 Ribosomal protein L12

CAA48183

AVEAPEKIEKIGSEISSLTLEEARDEA EEAKKTLEEAGAKVSIA

48.13

19,698.54 5.51

LHB1B2 (Photosystem II NP_565786 light harvesting complex gene 1.5)

F-box family protein

IIGESLDLIKPPSLFDGTTR

30,746

WVLDWIQNVLKR

a

Minus and positive values of the Av. ratios indicate down- and up-regulation of proteins upon formaldehyde exposure, respectively

b

Only part of the peptide sequences obtained are listed

irradiance (Romanowska et al. 2008). In addition, ribulose1,5-bisphosphate carboxylase/oxygenase, the first enzymes of the Calvin cycle in the carbon fixation process, was also identified consistently as up-regulated protein upon both VOC treatments, although the light condition was not sufficiently intense as to increase photosynthetic rate or high light stimuli. Therefore, the induced expression of ATP synthase CF1 possibly suggests that plants exposed to formaldehyde and toluene might operate similar responses with high light stimulus to escape from the toxic environment, probably by increasing their carbohydrate metabolism for more effective utilization of the carbon atoms in the formaldehyde molecules as a carbon source. Such a

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response would explain how indoor plants can remove the toxic VOCs under insufficient light condition. Interestingly, glutathione transferase was significantly up-regulated upon toluene treatment (Table 3), but not upon the formaldehyde. Glutathione transferases are multifunctional proteins encoded by a large gene family and have been demonstrated to be involved in the toxin catabolic process and the herbicide detoxification process, as well as in the oxidative stress tolerance (Edwards et al. 2000). Interestingly, In2-1 protein identified as up-regulated protein also contains a domain for glutathione transferase activity, implying a possible role for the detoxification process in A. thaliana upon toluene

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treatment. Glutathione transferase was previously reported to be induced by ozone treatment in A. thaliana (Sharma and Davis 1994). Therefore, glutathione transferase seems to be involved in the detoxification process under the oxidative stress caused by toluene and ozone, which might explain why the plant exposed to the toluene looks much healthier than that of formaldehyde. Like other abiotic stress responses, the VOC-mediated process is possibly linked to be the hormonal-mediated stress responses as ethylene-responsive transcriptional factor which was identified as a toluene-induced protein. Although strong molecular evidence is not yet available, ethylene has been regarded for a long time as the best candidate for the plant hormones that might play an essential role in the stress response induced by toxic VOCs in plants (Cape 2003). Therefore, the identification via proteome analysis in this study of an ethylene-responsive transcription factor as one of the toluene-responsive proteins is very meaningful, although its molecular mechanism on the VOC-mediated plant response is largely unknown. Not only to the ethylene but also the jasmonic acid was also implicated to be involved in the detoxification of formaldehyde in plants as the transcription level of formaldehyde dehydrogenase was demonstrated to be dependent on the jasmonic acid in plants (Sandermann 2004; Dıáz et al. 2003). Furthermore, the application of jasmonic acid to the leaves of Quercus ilex has been reported to decrease the uptake of exogenously treated formaldehyde (Filella et al. 2006), implying an essential role of jasmonic acid in VOCmediated responses in A. thaliana. Further characterization of ethylene and jasmonic acid in VOC-mediated response need to be carried out whether or not they crosstalk or act independently for the VOC-mediated response in plants. Many researchers have considered the role of plants in removing indoor air contamination to be very controversial, especially in the indoor condition, mainly due to the insufficient light level for active gas exchange during photosynthesis. Thus, the potential role of plants in VOC detoxification has often been neglected in the indoor environment, compared to those of root-associated microorganisms (Wood et al. 2002). However, our proteome analysis using 2D-DIGE of VOC-exposed A. thaliana has supported the suggestion that plants can actively detoxify VOCs even in a low light environment, probably via accelerated photosynthetic apparatus by activating carbon fixation and metabolism, as well as via stress responses mediated by ethylene and/or jasmonic acid. Since many of the VOC-inducible proteins identified in these experiments have not been functionally characterized at the molecular level, further characterization of these VOC-responsive proteins may provide better knowledge of the VOC-mediated plant response in plants.

1613 Acknowledgments We thank Dr. Jongsoo Chang (Korea National Open University) for technical support in 2D-DIGE analysis. We also thank Dr. Sang-Bong Choi (Department of Biological Sciences at Myungee University) for the permission to use fluorescence microscopic facility. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-531-F00005).

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