Proteomic Analysis of Arabidopsis Glutathione S-transferases from Benoxacor- and Copper-treated Seedlings
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m402807200
ISSN1083-351X
AutoresAaron P. Smith, Ben P. DeRidder, Woei‐Jiun Guo, Erin H. Seeley, Fred E. Regnier, Peter B. Goldsbrough,
Tópico(s)Agriculture and Biological Studies
ResumoGlutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions. Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions. Glutathione S-transferases (GSTs) 1The abbreviations used are: GST, glutathione S-transferase; DHAR, dehydroascorbate reductase; MS, mass spectrometry; MS/MS, tandem mass spectrometry. (also glutathione transferases) are a collection of multifunctional proteins that are found in essentially all organisms. In addition to their well known role as phase II detoxification enzymes (1Coleman J.O.D. Blake-Kalff M.M.A. Davies T.G.E. Trends Plant Sci. 1997; 2: 144-151Abstract Full Text PDF Scopus (528) Google Scholar), GSTs also provide protection against oxidative stress, catalyze various metabolic reactions, and serve as carrier proteins for a number of endogenous ligands (2Marrs K.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 127-158Crossref PubMed Scopus (1112) Google Scholar, 3Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1462) Google Scholar, 4Dixon D.P. Cole D.J. Edwards R. Arch. Biochem. Biophys. 2000; 384: 407-412Crossref PubMed Scopus (67) Google Scholar, 5Dixon D.P. Davis B.G. Edwards R. J. Biol. Chem. 2002; 277: 30859-30869Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). GSTs are grouped into distinct classes based on a number of criteria, including protein sequence, gene structure, immunological cross-reactivity, kinetic properties, and structural characteristics (3Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1462) Google Scholar). Some classes of GSTs are taxa-specific whereas others are found across kingdoms. Plant GSTs fall into one of six classes: Phi, Tau, Theta, Zeta, Lambda, and glutathione-dependent dehydroascorbate reductases (DHARs). The two largest classes, Phi and Tau, are plant-specific, whereas the Theta and Zeta classes are also found in mammals, fungi, and insects. To date, Lambda and DHAR-type GSTs have been identified only in plants (5Dixon D.P. Davis B.G. Edwards R. J. Biol. Chem. 2002; 277: 30859-30869Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Many plant GSTs have been characterized as enzymes that catalyze the conjugation of glutathione (GSH) to a variety of xenobiotics, notably herbicides. Studies with compounds known as herbicide safeners, which are used to enhance herbicide tolerance of crop plants, have contributed to the understanding of this primary function of GSTs in plants (6Davies J. Caseley J.C. Pestic. Sci. 1999; 55: 1043-1058Crossref Google Scholar). Safeners may increase herbicide tolerance by a number of mechanisms, but the bulk of the evidence indicates they selectively induce GSTs that detoxify specific herbicides. For example, the safener benoxacor protects maize from chloroacetanilide herbicides (e.g. metolachlor and alachlor) by increasing the expression of GSTs that efficiently conjugate these herbicides to GSH but has no protective effect on dicotyledenous weeds (7Irzyk G.P. Fuerst E.P. Plant Physiol. 1993; 102: 803-810Crossref PubMed Scopus (110) Google Scholar). As potent inducers of GST expression, safeners can help to elucidate the signaling pathways that govern the expression of plant GSTs involved in xenobiotic detoxification. A recent study (8DeRidder B.P. Dixon D.P. Beussman D.J. Edwards R. Goldsbrough P.B. Plant Physiol. 2002; 130: 1497-1505Crossref PubMed Scopus (133) Google Scholar) demonstrated that RNA expression of several Arabidopsis GST genes is differentially induced by a number of safeners. These results suggest that induction of Arabidopsis GSTs by safeners is complex and occurs via multiple signaling pathways. Aside from their role in xenobiotic detoxification, plant GSTs also provide protection against oxidative stress. Functioning as glutathione peroxidases, plant GSTs can catalyze the reduction of hydroperoxides to less harmful alcohols (9Roxas V.P. Smith R.K. Allen E.R. Allen R.D. Nat. Biotechnol. 1997; 15: 988-991Crossref PubMed Scopus (475) Google Scholar, 10Cummins I. Cole D.J. Edwards R. Plant J. 1999; 18: 285-292Crossref PubMed Scopus (291) Google Scholar). Expression of a GST with glutathione peroxidase activity in transgenic tobacco provided protection against oxidative stress (11Roxas V.P. Lodhi S.A. Garrett D.K. Mahan J.R. Allen R.D. Plant Cell Physiol. 2000; 41: 1229-1234Crossref PubMed Scopus (397) Google Scholar). Plant GSTs may also protect against oxidative stress by conjugating GSH to toxins produced as a result of oxidative damage to endogenous compounds. For example, heavy metals (e.g. copper) can induce oxidative stress, resulting in membrane lipid peroxidation and the formation of cytotoxic alkenals (12De Vos C.H.R. ten Bookum W.M. Vooijs R. Schat H. de Kok L.J. Plant Physiol. Biochem. 1993; 31: 151-158Google Scholar). One such alkenal, 4-hydroxynonenal, is a substrate for Phi class GSTs from sorghum (13Gronwald J.W. Plaisance K.L. Plant Physiol. 1998; 117: 877-892Crossref PubMed Scopus (108) Google Scholar), and wheat Tau class GSTs exhibit similar activities (14Cummins I. Cole D.J. Edwards R. Pestic. Biochem. Physiol. 1997; 59: 35-49Crossref Scopus (102) Google Scholar). In addition to their roles in xenobiotic detoxification and oxidative stress tolerance, plant GSTs also catalyze metabolic isomerization reactions. For example, AtGSTZ1 participates in tyrosine catabolism in Arabidopsis by catalyzing the cis-trans conversion of maleylacetoacetate to fumarylacetoacetate (4Dixon D.P. Cole D.J. Edwards R. Arch. Biochem. Biophys. 2000; 384: 407-412Crossref PubMed Scopus (67) Google Scholar). Finally, some plant GSTs function as nonenzymatic binding proteins of endogenous compounds such as anthocyanins and phytohormones (2Marrs K.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 127-158Crossref PubMed Scopus (1112) Google Scholar, 15Mueller L.A. Goodman C.D. Silady R.A. Walbot V. Plant Physiol. 2000; 123: 1561-1570Crossref PubMed Scopus (340) Google Scholar, 16Gonneau J. Mornet R. Laloue M. Physiol. Plant. 1998; 103: 114-124Crossref Scopus (49) Google Scholar). The GST gene family in Arabidopsis includes 53 genes (5Dixon D.P. Davis B.G. Edwards R. J. Biol. Chem. 2002; 277: 30859-30869Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 17Wagner U. Edwards R. Dixon D.P. Mauch F. Plant Mol. Biol. 2002; 49: 515-532Crossref PubMed Scopus (422) Google Scholar). Given the diverse functions of GSTs in plants, it is not surprising that a wide range of factors induce RNA expression of these genes, including herbicides, heavy metals, extreme temperatures, phytohormones, and pathogen attack (2Marrs K.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 127-158Crossref PubMed Scopus (1112) Google Scholar, 3Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1462) Google Scholar). Although an abundance of data has been gathered on RNA expression of many of these GSTs, much less is known about the regulation of GST protein expression. The objectives of this study, therefore, were to identify the GSTs expressed in Arabidopsis seedlings and to quantify the impact of various environmental conditions on GST protein expression. Two major functions carried out by plant GSTs are detoxification of xenobiotics and protection against oxidative stress. Therefore, we examined the expression of Arabidopsis GSTs in response to an herbicide safener, benoxacor, and an inducer of oxidative stress, copper, both of which are agents that provide conditions for addressing these two important functions of plant GSTs. Because of the paucity of information about GST protein expression, a proteomic approach was used to examine multiple GST proteins simultaneously. Using a method adapted from the signature peptide approach (18Geng M. Ji J. Regnier F.E. J. Chromatogr. A. 2000; 870: 295-313Crossref PubMed Scopus (146) Google Scholar, 19Regnier F. Amini A. Chakraborty A. Geng M. Ji J. Riggs L. Sioma C. Wang S. Zhang X. LC GC North America. 2001; 19: 200-213Google Scholar), eight GSTs were identified in Arabidopsis seedlings: AtGSTF2, AtGSTF6, AtGSTF7, AtGSTF8, AtGSTF9, AtGSTF10, AtGSTU19, and AtGSTU20. The herbicide safener benoxacor induced accumulation of AtGSTU19, whereas copper treatment resulted in higher levels of AtGSTF2, AtGSTF6, AtGSTF7, and to a lesser extent AtGSTU19. In contrast, AtGSTF9 was significantly less abundant in response to both treatments. Although RNA expression generally reflected the changes in abundance of GST proteins in response to benoxacor and copper, transcript levels of AtGSTF8, AtGSTF9, and AtGSTF10 were notable exceptions. Protein Extraction and Purification—Seeds of Arabidopsis thaliana, ecotype Columbia, were germinated in half-strength Murashige and Skoog liquid medium, grown for 9 days, and treated for 24 h with 100 μm benoxacor (Syngenta, Greensboro, NC) or 50 μm CuSO4. The seedlings were then harvested, weighed, frozen in liquid nitrogen, and stored at –70 °C. Seedlings (75–100 g) were ground to a fine powder in liquid nitrogen using a chilled mortar and pestle and homogenized in 3 volumes (w:v) of extraction buffer (50 mm Tris-HCl, pH 8, 5 mm dithiothreitol, 1 mm EDTA) using a Cyclone IQ2 microprocessor system (Virtis, Gardiner, NY). After filtration through eight layers of cheesecloth and centrifugation at 12,000 × g for 15 min, the supernatant was brought to 80% saturation with solid ammonium sulfate ((NH4)2SO4), and centrifuged at 12,000 × g for 20 min. The pellets were resuspended in a small volume of saturated (NH4)2SO4 and stored at –20 °C. The precipitated protein was centrifuged at 12,000 × g for 10 min and then dissolved in ∼2 volumes of phosphate-buffered saline buffer containing 5 mm dithiothreitol. Protein samples were dialyzed (12,000–14,000 molecular weight cutoff) (Spectra/Por, Spectrum Laboratories Inc., Rancho Dominguez, CA) for 2 h against 1 liter of phosphate-buffered saline containing dithiothreitol, with one change of buffer. The samples were centrifuged at 12,000 × g for 20 min to remove undissolved material. The proteins were applied to a GSH-agarose affinity column (5-ml bed volume) (G-4510, Sigma) at a flow rate of 0.5 ml min–1 followed by a wash (5 column volumes) with phosphate-buffered saline buffer at a flow rate of 1 ml min–1. Bound proteins were eluted with 20 ml of elution buffer (50 mm Tris-HCl, pH 7.5, 10 mm GSH) at a flow rate of 1 ml min–1. Eluted proteins were concentrated and dialyzed against 0.1 m HEPES buffer, pH 8.0, using Amicon Ultra centrifugal filter devices (10-kDa cutoff) (Millipore Corp., Bedford, MA). Protein samples were kept at 4 °C or on ice at all times, and protein concentrations were determined using bovine serum albumin as a standard (20Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222135) Google Scholar). Immunoblot analyses were carried out as described using polyclonal antibodies against AtGSTF2 or AtGSTU19 (21Smith A.P. Nourizadeh S.D. Peer W.A. Xu J. Bandyopadhyay A. Murphy A.S. Goldsbrough P.B. Plant J. 2003; 36: 433-442Crossref PubMed Scopus (114) Google Scholar). Trypsin Digestion and Peptide Acetylation—Protein samples obtained by GSH affinity chromatography were diluted with 100 mm HEPES buffer, pH 8.0, containing 9 m urea to give a final urea concentration of 7 to 8 m. Cysteine sulfhydryls were reduced by the addition of 10 mm dithiothreitol followed by incubation at 37 °C for 2 h. Free sulfhydryl groups were alkylated with iodoacetic acid (final concentration 20 mm) in the dark on ice for 2 h. The alkylation reaction was quenched by the addition of 10 mm cysteine followed by incubation on ice for 5 min. Samples were diluted with 0.1 m HEPES, pH 8.0, to a final urea concentration of 1 m. One microgram of sequencing grade-modified trypsin (Promega, Madison, WI) was added for every 50 μg of protein in the sample. Proteolysis was carried out at 37 °C for 12 h and then quenched with 1 mg of Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK). One milligram of acetoxysuccinamide was added to the control samples, and 1 mg of d3-acetoxysuccinamide, synthesized as described previously (18Geng M. Ji J. Regnier F.E. J. Chromatogr. A. 2000; 870: 295-313Crossref PubMed Scopus (146) Google Scholar), was added to the experimental samples. The samples were stirred at room temperature for 4 h, and each experimental sample (derived from either benoxacor- or copper-treated tissue) was combined with its respective control sample. To reverse the acetylation of hydroxyl groups on tyrosine residues, 2 mg of hydroxylamine hydrochloride was added to each mixture. The pH was adjusted to 12.0 with 5 m NaOH, and the mixtures were stirred at room temperature for 10 min. Glacial acetic acid was added to adjust the pH to 8.0, and the samples were dried in a centrifuge dryer. Reverse-phase Liquid Chromatography and Mass Spectrometry—The acetylated tryptic peptides were dissolved in 500 μl of water and separated over a Microsorb-MV 100–5 C18 reverse-phase column (Varian, Palo Alto, CA), with water and acetonitrile (both containing 0.1% trifluoroacetic acid. Peptides were eluted at a flow rate of 1 ml min–1 using a gradient of 15–60% acetonitrile over 25 min followed by a step elution to 100% acetonitrile over 5 min. Thirty fractions of 1 ml each were collected, dried in a centrifuge dryer, and dissolved in 100 μl of 50% (v:v) methanol:water containing 0.1% trifluoroacetic acid. These samples were injected into a Q-Star electrospray ionization quadrupole time-of-flight mass spectrometer (Perseptive Biosystems Inc., Framingham, MA) at 8 μl min–1. Masses were scanned from m/z 300 to 2000 at default voltage potentials, and abundant peptides were fragmented by restricting the ion gate voltage. Peptide parent ion masses were analyzed with Mascot (www.matrixscience.com). Mascot parameters included proteolysis by trypsin/chymotrypsin with one missed cleavage, alkylation of cysteine residues with iodoacetic acid, and size tolerances of 0.5 Da for peptides and 0.2 Da for peptide fragments. Variable modifications included sodiated (C-terminal), sodiated (D/E), acetyl heavy (K), acetyl heavy (N-terminal), acetyl light (K), and acetyl light (N-terminal). The relative abundance of GST peptides in control and benoxacor- or copper-treated samples was calculated as the ratio of their respective monoisotopic peak intensities. Isolation and Analysis of RNA—Total RNA was isolated from Arabidopsis seedlings, separated by electrophoresis through formaldehyde-agarose gels, transferred to a nylon membrane, UV cross-linked, and hybridized with 32P-labeled cDNAs as described previously (21Smith A.P. Nourizadeh S.D. Peer W.A. Xu J. Bandyopadhyay A. Murphy A.S. Goldsbrough P.B. Plant J. 2003; 36: 433-442Crossref PubMed Scopus (114) Google Scholar). For real-time PCR analysis of the transcript for the copper-induced protein encoded by At2g28540, RNA was first treated with DNase (DNA-free, Ambion, Austin, TX). Complementary DNA was synthesized in a 30-μl reaction containing 2 μg of RNA, 500 ng of oligo(dT), 0.5 mm dNTPs, and 1 μl of avian myeloblastosis virus reverse transcriptase (Promega). After incubation at 42 °C for 50 min, the reverse transcriptase was inactivated by incubation at 70 °C for 10 min. The cDNA was diluted to 600 μl, and 5 μl was used as a template for PCR. The gene-specific primers for At2g28540 were 5′-ACCACCTGGCTTTTCAGTT-3′ and 5′-TCTGCATAGACAAAGGGTT-3′. Expression levels were normalized to that of a tubulin RNA (At5g12250) using the primers 5′-TGGGAACTCTGCTCATATCT-3′ and 5′-GAAAGGAATGAGGTTCACTG-3′. Real-time PCR was performed using an iCycler iQ (Bio-Rad) with SYBR I as the fluorescence dye. Each reaction was performed in 20-μl aliquots containing Taq DNA polymerase, the appropriate primer pair (250 nm), 0.25 mm dNTPs, 2 mm MgCl2, SYBR I, and fluorescein. After the initial denaturation at 95 °C for 3 min, 40 cycles of denaturation (95 °C, 30 s), annealing (55 °C, 20 s), and polymerization (72 °C, 20 s) were performed. All assays were carried out in triplicate. The PCR products were quantified at each amplification step. The specificity of each PCR reaction was assessed by agarose gel electrophoresis and melting curve analysis, which was determined by measuring the decline in fluorescence as temperature increased from 55 °C to 95 °C. Threshold cycles (CT) at which the fluorescence of the PCR product-SYBR I complex first exceeded the background were determined by integrated analysis software. To compare the level of expression in different samples, the difference between the CT of the target gene and the CT for the tubulin standard was calculated as ΔCT (target gene) = CT (target) – CT (tubulin). Relative transcript levels were calculated as 1000 × 2ΔCT. Identification of Expressed Arabidopsis GSTs—Proteolysis of a soluble protein extract from seedlings generates a complex mixture of a very large number of peptides, which can pose problems for further analysis. To reduce this complexity and to preferentially examine GSH-binding proteins such as GSTs, a GSH affinity purification step was included before proteolysis. Proteins eluted from the GSH affinity column were reduced, alkylated, and digested with trypsin. The resulting peptides were acetylated and analyzed by mass spectrometry (MS). In our initial experiments, these peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight MS. Although nearly 100 peptides were detected, they could not be assigned unequivocally to specific Arabidopsis GSTs because the masses of most peptides matched the predicted masses of peptides from more than one Arabidopsis GST (data not shown). Therefore, tandem mass spectrometry (MS/MS) was used to obtain information about the amino acid sequences of peptides, which allowed for unambiguous identification of the specific GSTs from which most of these peptides were derived. This approach was used to analyze and identify the GST proteins expressed in Arabidopsis seedlings. The recovery of proteins after GSH affinity chromatography indicates that GSTs comprise ∼0.1% of the soluble protein in Arabidopsis seedlings. A total of 87 peptides obtained from this affinity-purified sample were analyzed by MS/MS. Based on high probability matches obtained by using the Mascot peptide identification program, 38 of these peptides were identified as deriving from one of eight Arabidopsis GSTs: AtGSTF2, AtGSTF6, AtGSTF7, AtGSTF8, AtGSTF9, AtGSTF10, AtGSTU19, or AtGSTU20 (Table I). At least three unique peptides were detected for each of these GSTs except for AtGSTU20, which was represented by a single peptide. Another 24 peptides were identified as being derived from Arabidopsis GSTs, but similarities among the members of this protein family prevented these peptides from being unequivocally assigned to a single GST. Consequently, these peptides were excluded from further analyses. For 22 peptides, the MS/MS spectra did not give a significant match with any protein in the data base (data not shown). Only one peptide (IQNGCSNVVSVDADSVVDGY) was identified that had a significant match to a protein other than an Arabidopsis GST. The function of this protein, encoded by gene At2g28540, is unknown and does not share significant sequence homology with any other characterized gene product. These results demonstrate that this technique can be used to identify GST proteins expressed in Arabidopsis seedlings and that GSH affinity chromatography provides an efficient method to reduce the complexity of protein samples, allowing for analysis focused on GSTs.Table 1Peptides identified in samples derived from untreated, copper-treated, and benoxacor-treated Arabidopsis seedlingsGSTm/zChargePeptide sequenceRelative expressionCopperBenoxacorGSTF2540.32LAFEQIFK2.611.11599.32VFGHPASIATR1.891.21757.73AIMAIGMQVEDHQFDPVASK5.021.26847.92YENQGTNLLQTDSK3.340.93959.43NISQYAIMAIGMQVEDHQFDPVASK2.461.151005.02SIYGLTTDEAVVAEEEAK4.880.891033.23YLAGETFTLTDLHHIPAIQYLLGTPTK3.281.14GSTF6780.42NVDFEFVHVELK5.211.25803.92AITQYIAHEFSDK3.701.07987.51GNNLLSTGK3.061.10GSTF7543.82GNQLVSLGSK11.751.07780.41YLASDK8.001.14794.42NLDFEFVHIELK10.22ND850.41EPFIFR9.671.25GSTF8677.31LAFER1.311.21759.42DLQFELIPVDMR1.521.40799.51VIDLQK1.380.92845.41VSEWIK1.261.141020.51VLATLYEK1.161.101159.02ATTNVWLQVEGQQFDPNASK1.271.27GSTF9752.32GVAFETIPVDLMK0.470.29799.92LAGVLDVYEAHLSK0.360.29970.51ALVTLIEK0.490.29992.83YLAGDFVSLADLAHLPFTDYLVGPIGK0.62NDGSTF10601.82IPVLVDGDYK0.790.71620.31YSLPV0.940.91737.82QPEYLAIQPFGK0.940.81747.42VLTIYAPLFASSK1.020.84831.52LAEVLDVYEAQLSK0.840.70942.51AVVTLVEK0.890.71GSTU19561.32VTEFVSELR1.566.24663.82NPILPSDPYLR1.736.13726.32FANFSIESEVPK1.845.74737.92SPLLLQMNPIHK1.486.12858.41KVWATK1.616.43873.42SLPDPEKND5.86961.51DFIEILK1.266.75GSTU20715.92SPLLLQSNPIHK0.770.50 Open table in a new tab Altered Expression of GSTs in Response to Copper and Benoxacor—The RNA expression of plant GSTs is influenced by many factors, but it is not known whether the expression of GST proteins parallels transcript accumulation under all these conditions. By combining MS-based analysis of peptides with differential isotopic labeling, it is possible to quantify changes in protein expression (19Regnier F. Amini A. Chakraborty A. Geng M. Ji J. Riggs L. Sioma C. Wang S. Zhang X. LC GC North America. 2001; 19: 200-213Google Scholar). In our experiments, GSH affinity-purified proteins were digested with trypsin and labeled with either acetate (control) or trideuteroacetate (treated) before being combined and analyzed by MS/MS. Because the labeling reaction targets primary amino groups, the amino terminus of every peptide was acetylated. In addition, if the peptide contained a lysine, this residue was labeled on its ϵ-amino group. Peptides that contain only a single amino-terminal primary amine appear in the mass spectrum as a pair of peaks, i.e. a doublet separated by three mass units, with one peak from the control sample and the other from the treated sample (Fig. 1, a and b). Singly charged peptides that also contain a lysine residue appear as doublets separated by six mass units; however, most peptides derived from Arabidopsis GSTs were ionized with two protons (i.e. doubly charged). Therefore, a lysine-containing doubly charged peptide appears as a doublet separated by three mass units (Fig. 1, c and d). Differences in expression between control and treated samples of specific GSTs were determined by comparing the relative abundance of the monoisotopic peaks of peptides derived from each protein. This technique was used to analyze the influence of copper and benoxacor on Arabidopsis GST protein expression. Specifically, two independent experiments were carried out by combining a control sample derived from untreated Arabidopsis seedlings with an experimental sample derived from either copper- or benoxacor-treated Arabidopsis seedlings; for example, the abundance of a peptide derived from AtGSTF7 was nearly 10-fold higher in copper-treated seedlings (Fig. 1a), whereas benoxacor had essentially no effect (Fig. 1b). In contrast, a peptide derived from AtGSTU19 was ∼6-fold more abundant in benoxacor-treated seedlings (Fig. 1d) but increased only 2-fold after copper treatment (Fig. 1c). With few exceptions, the peptides detected in samples from both copper- and benoxacor-treated seedlings were the same as those observed in control tissues. Therefore, GSTs that were not expressed under control conditions remained silent (or below the limit of detection of these experiments) after seedlings were treated with copper or benoxacor. The relative expression of each peptide in response to copper and benoxacor was calculated based on the monoisotopic peak intensities of each doublet (Table I). Seven of the eight GSTs that were expressed were represented by three or more peptides. Changes in protein expression of these seven GSTs were estimated by averaging the relative abundance of all peptides derived from each protein (Fig. 2). Individual peptides derived from the same GST generally showed very similar changes in abundance within a treatment, providing some internal validation of this approach. Copper increased the abundance of three GSTs by at least 3-fold, and AtGSTF7 was induced ∼10-fold. AtGSTU19 also was somewhat more abundant in copper-treated seedlings. In contrast, benoxacor strongly induced AtGSTU19, as previously documented (8DeRidder B.P. Dixon D.P. Beussman D.J. Edwards R. Goldsbrough P.B. Plant Physiol. 2002; 130: 1497-1505Crossref PubMed Scopus (133) Google Scholar), but no other GSTs were significantly more abundant after treatment with this herbicide safener. Neither copper nor benoxacor had a pronounced effect on expression of AtGSTF8 or AtGSTF10, but the abundance of AtGSTF9 was reduced more than 50% by both treatments. A similar reduction in abundance of AtGSTU20 was observed, although this was based on analysis of a single peptide derived from this protein (Table I). Immunoblot Analysis of AtGSTU19 and AtGSTF2—Proteomic analysis of Arabidopsis GSTs purified from copper- and benoxacor-treated seedlings demonstrated differential expression of several Arabidopsis GSTs including AtGSTU19 and AtGSTF2. To corroborate these data, immunoblot analyses were carried out using antibodies raised against recombinant forms of AtGSTU19 and AtGSTF2. AtGSTU19 protein expression was induced in response to benoxacor and to a lesser extent to copper, whereas AtGSTF2 expression was induced by copper but was unaffected by benoxacor (Fig. 3). Immunoblot analyses of the effects of copper and benoxacor on expression of AtGSTU19 and AtGSTF2 produced results similar to those obtained in the proteomic analyses described above. Anti-AtGSTF2 antibodies detected an additional protein with a slightly lower molecular weight than that of AtGSTF2 (Fig. 3). Similar results have been obtained previously (21Smith A.P. Nourizadeh S.D. Peer W.A. Xu J. Bandyopadhyay A. Murphy A.S. Goldsbrough P.B. Plant J. 2003; 36: 433-442Crossref PubMed Scopus (114) Google Scholar), but the nature of this smaller protein has not been investigated further. Expression of Arabidopsis GST RNAs in Response to Copper and Benoxacor—RNA blot analysis was used to compare the RNA expression of the seven abundantly expressed GSTs identified in the proteomic analyses with their protein accumulation patterns. Copper treatment increased the RNA expression of AtGSTF2, AtGSTF6, AtGSTF7, and to a lesser extent, AtGSTU19, whereas benoxacor induced the RNA expression of AtGSTU19 (Fig. 4). These results correspond with the observed changes in abundance of these GST proteins under the same conditions. However, RNA expression of AtGSTF10 and AtG-STF8 was induced by copper and benoxacor, respectively (Fig. 4) although protein expression experiments showed that the abundance of AtGSTF10 and AtGSTF8 was not affected by either treatment (Fig. 2). In addition, neither copper nor benoxacor affected RNA expression of AtGSTF9 (Fig. 4), even though AtGSTF9 protein expression was down-regulated in response to these stimuli (Fig. 2). RNA expression of At2g28540, the gene encoding the non-GST protein that was induced by copper, was also examined. Because this transcript could not be detected by blot hybridization, real-time reverse transcriptase PCR was used. The transcript of At2g28540 was ∼5-fold more abundant in copper-treated seedlings (ΔCT of –1.25 ± 0.1) than in control seedlings (ΔCT of –3.60 ± 0.5), relative to the expression of control tubulin RNA. RNA expression of genes encoding GSTs is influenced by diverse abiotic and biotic factors in plants and other organisms, but relatively little is known about the effects of these stimuli on expression
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