Proteome Approach To Characterize Proteins Induced by Antagonist Yeast and Salicylic Acid in Peach Fruit Zhulong Chan,†,‡ Guozheng Qin,† Xiangbin Xu,†,‡ Boqiang Li,† and Shiping Tian*,† Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, and Graduate School of Chinese Academy of Sciences, Beijing 100093, China Received September 15, 2006
Proteins induced by antagonist yeast Pichia membranefaciens and salicylic acid (SA) in peach fruit were determined using proteome analysis in this study. Both the yeast and SA enhanced the resistance of peach fruit and delayed the initiation infection of Penicillium expansum. When quadrupole timeof-flight tandem mass spectrometer was used, a total of 25 proteins could be identified as significantly up- or down-regulated in response to at least one activitor. According to the function, these proteins were attributed to protein metabolism, defense response, transcription, energy metabolism, and cell structure. Among them, 6 antioxidant and 3 pathogenesis-related (PR) proteins were induced by P. membranefaciens or SA treatments. The induction results of these proteins were related to treatment time. Six other proteins were identified as the enzymes which catalyze the reactions of glycolysis and tricarboxylic acid cycle. In addition, both the yeast and SA treatments enhanced the transcript and translation expression of the catalase gene. These results suggested that antioxidant and PR proteins, as well as enzymes associated with sugar metabolism, were involved in resistance of peach fruit induced by P. membranefaciens and SA. Keywords: antagonist yeast • induced resistance • peach • proteome • salicylic acid
Introduction Plants protect themselves from disease-causing organisms by activating a broad array of defense responses that ultimately inhibit growth and spread of invading pathogens. The response involves the specific recognition of the pathogens and development of a resistance to protect the plant from further attacks by the pathogens.1-3 This phenomenon is known as induced resistance (IR) and can be triggered by a variety of biotic and abiotic activitors.4,5 In general, two types of IR are characterized according to the reviews by Hammerschmidt6 and Welling:7 one is systemic acquired resistance (SAR) and the other is induced systemic resistance (ISR). Salicylic acid (SA) has emerged as a key signaling component involved in the activation of certain plant defense responses.8 Exogenous application of SA protects plants against certain pathogens and activates SAR in a wide variety of plant species,9 including harvested fruit.10 Antagonist yeast could effectively control postharvest diseases in various fruits.11-13 One of the mechanisms was considered to induce several biochemical defenses of fruit against fungal pathogens.14-16 The results of our previous study demonstrated that antagonist Pichia membranefaciens and SA treatments could stimulate synthesis of antioxidant enzymes in sweet cherry fruit.17 Therefore, induced resistance is a very attractive strategy for controlling diseases * To whom correspondence should be addressed. Tel: +86-10-6283-6559. Fax: +86-10- 8259-4675. E-mail:
[email protected]. † Institute of Botany, Chinese Academy of Sciences. ‡ Graduate School of Chinese Academy of Sciences. 10.1021/pr060483r CCC: $37.00
2007 American Chemical Society
in harvested fruit. However, the mechanisms of induced resistance remain an enigma and are still far from understood. As an effective strategy for protein analysis, two-dimensional gel electrophoresis (2-DE) has been used successfully to identify novel protein components in plant in response to cold, salt, fungi, bacteria, and virus.18-22 Although some proteins differentially expressed in a colorless strawberry mutant23 or associated with maturity of fruit24,25 were recently detected using a 2-DE-based proteomics approach, little information about defense response of harvested fruit was reported using proteomics approach. In the present study, induced resistance on peach fruit against blue mold rot caused by Penicillium expansum and proteins induced by yeast antagonist P. membranefaciens and SA were analyzed using a proteomics approach. Defenserelated proteins and other proteins involved in sugar metabolism were induced after P. membranefaciens and SA treatments. The transcript and translation levels of the catalase gene were also compared. Additionally, the defense mechanisms of peach fruit induced by the yeast and SA were further discussed.
Materials and Methods Fruit Samples and Microbial Agents. Peach fruit (Prunus persica L. Batsch) were harvested at commercial maturity from an experimental orchard in the Pinggu district of Beijing, China, and immediately transported to our laboratory where they were sorted based on size and the absence of physical injuries or infections. The individual fruit was about 250 ( 20 g in weight. Selected fruit were surface-disinfected with 2% (w/v) sodium Journal of Proteome Research 2007, 6, 1677-1688
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Table 1. Peach Fruit Treated with Antagonist Yeast and SA groups
treatments
analysis
1
Treated with water and then inoculated with P. expansum Treated with yeast and then inoculated with P. expansum Treated with SA and then inoculated with P. expansum Treated with water without pathogen inoculation Treated with yeast without pathogen inoculation Treated with SA without pathogen inoculation
For disease development investigation
2
hypochlorite for 2 min, washed with sterile water, and air-dried prior to use. Antagonist yeast P. membranefaciens Hansen was isolated from wounds of peach fruit26 following the method of Wilson and Chalutz27 and identified by CABI Bioscience Identification Services (International Mycological Institute, U.K.). The yeast was grown in 250 mL conical flasks containing 50 mL of nutrient yeast dextrose broth (NYDB: 1 g of beef extract, 10 g of glucose, 5 g of soya peptone, 5 g of NaCl, and 5 g of yeast extract in 1000 mL of water) on a rotary shaker at 200 rpm for 48 h at 28 °C. Yeast cells were centrifuged at 3944g (SORVALL Biofuge Stratos, Germany) for 10 min, resuspended in sterile, distilled water, and adjusted to a concentration of 1 × 108 cells mL-1 with a haemocytometer. P. expansum Link, a pathogenic fungus causing serious decay of harvested peach fruit during storage periods, was isolated from infected peach fruit with a typical symptom of blue mold. The fungus was maintained on potato dextrose agar (PDA) at 4 °C. Spores of P. expansum from 14-day-old cultures at 25 °C were obtained by flooding the cultures with sterile, distilled water containing 0.05% (v/v) Tween-80. The spore suspension was filtered through four layers of sterile cheesecloth. The concentration of spores was adjusted to 1 × 104 spores mL-1 with the aid of a haemocytometer. Treatments with Antagonist and SA. Peach fruit were immersed in the yeast cell suspension at 1 × 108 cells mL-1 or SA (Sigma) solution at a concentration of 0.5 mM for 10 min.10 Fruit immersed with sterile, distilled water were used as the control. All fruits were then air-dried at 25 °C for 2 h and divided into two groups. The treatment details of different groups are listed in Table 1. For the first group, a uniform wound (4 mm deep, 3 mm wide wound) was made at the equator of the fruit using a sterile nail. Aliquots of 10 µL suspension of P. expansum at 1 × 104 spores mL-1 were inoculated into each wound site. Fruit were put into 200 mm × 130 mm × 50 mm plastic boxes with plastic film to maintain a high relative humidity (95%) and stored at 25 °C. Disease incidence and lesion diameter were measured daily after treatment according to the following formulas: Disease incidence (%) )
∑ Number of decayed peach fruit
Total number of fruit in the treatment
× 100
Lesion diameter (cm) )
∑ Lesion diameter of decayed peach fruit Total number of fruit in the treatment
For the second group, fruit were treated with water, antagonist yeast, and SA, respectively, and then directly put into plastic boxes as described above without pathogen inoculation. At various time intervals after treatments, samples from fruit flesh were obtained for the following assays. All fruits were discarded after sampling. Three replications for each treatment 1678
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For other assays
were performed, and each replicate contained 15 fruits. The entire experiment was repeated twice. Assay of Fruit Quality Parameter. Soluble solids content (SSC) of peach fruit was determined using the same methods as previously reported.26 Flesh firmness (using 10 fruit) was determined on opposite peeled cheeks of the fruit using a Fruit Firmness Tester (FT-327, Italy), equipped with an 8-mm plunger tip. There were three replicates for each analysis per treatment. Protein Extraction from Fruit. The extraction of total proteins was performed as described by Shen et al. with some modifications.27 All procedures described below were carried out at 4 °C. Briefly, 4 g of flesh from 10 fruits was excavated using a sampler (5 mm deep and 7 mm in diameter) and mixed at 1, 2, 4, and 6 days after treatments. Samples were ground in liquid nitrogen and then homogenized in 1 mL of homogenization buffer containing 20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM EGTA, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 1% Triton X-100. The homogenate was centrifuged at 15 000g for 5 min, and the supernatant was removed to a new tube. The proteins in the supernatant were precipitated with 10% TCA on ice for 60 min, and then centrifuged at 15 000g for 10 min. The pellet was washed with acetone (twice with 10 mL and once with 1 mL) and then solubilized in 250 µL of lysis buffer containing 7 M urea, 2 M thiourea, 4% (v/v) CHAPS, 1% (w/v) DTT, 0.2% (v/v) Nonidet P40 (NP-40) (Sigma), and 2% (v/v) of a mixture of carrier ampholytes of pH 5-8 and pH 3.5-10 (Bio-Rad) in a ratio of 1:1. The protein concentration was determined according to Bradford’s method using bovine serum albumin as standard.28 The protein samples were stored at -70 °C prior to use. Two Dimensional-PAGE Analysis. Approximately 600 µg of protein samples were applied to 2-D PAGE. The first dimension electrophoresis was performed in a glass capillary tube (Daiichi pure Chemicals, Tokyo, Japan) of 13 cm length and 3 mm diameter according to the method described by Komatsu et al.29 Briefly, the gel solution contained 10% NP-40, 30% (w/v) acrylamide, 9.5 M urea, 10% ammonium persulfate, and an equal mixture of 2% carrier ampholytes (pH 3.5-10 and 5-8). Fifty microliters of 50% lysis buffer was used as the sample overlay buffer. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 800 V for 1 h. After the first-dimensional run, IEF gels were equilibrated in equilibration buffer (62.5 mM Tris-HCl, pH 6.8, 2.5% SDS, 10% (v/v) glycerol, and 5% 2-mercaptoethanol) for 15 min twice. Sodium dodecyl sulfate (SDS)-PAGE in the second dimension was performed with 15% separation gels and 5% stacking gels (175 mm × 200 mm × 1 mm) at a constant current of 30 mA. The gel was stained for 2 h with Coomassie brilliant blue (CBB) solution containing 500 mL of methanol, 150 mL of acetic acid, and 1 g of CBB R-250 in a 1000 mL volume. Gel Image Analyses. Stained gels were scanned immediately after destaining to minimize any possibility of fading. Gels were
Peach Fruit Proteome
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Figure 1. Development of blue mould in peach fruit after treatment with (a) sterile distilled water (control), (b) P. membranefaciens at 1 × 108 cells mL-1 (yeast), and (c) SA at 0.5 mM (SA) at 25 °C. (A) Symptoms of blue mold in peach fruit 6 days after inoculation; (B) disease incidence and lesion diameter of blue mold in peach fruit. Bars represent standard errors of the mean.
digitized using an ImageScanner (Amersham Biosciences) in transmission mode at a resolution of 600 dpi, and the data were analyzed with Image Master 2D Elite software (Amersham Pharmacia Biotech, Uppsala, Sweden) for three biological repeats for each treatment. The optimized parameters were as follows: saliency, 2.0; partial threshold, 5; and minimum area, 50. All gels from each treatment were matched to each other and to the other treatments, and spots were assigned arbitrary identifiers. The amount of a protein spot was expressed as the volume of that spot which was defined as the sum of the intensities of all the pixels that make up that spot. To correct the variability due to CBB staining and to reflect the quantitative variations of protein spots, the spot volumes were normalized as a percentage of the total volume in all of the spots in the gel. The protein spots with significant changes in intensity in a consistent direction (increase or decrease) were considered to be different. The identified protein spots were manually rechecked. In-Gel Tryptic Digestion. The CBB-stained protein spots were excised from the gel and destained twice with 50 mM NH4HCO3 in 50% (v/v) methanol for 1 h at 40 °C.30 Proteins were reduced with 10 mM DTT and 10 mM EDTA in 100 mM NH4HCO3 for 1 h at 60 °C and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at room temperature. The gel pieces were minced, allowed to dry, and then rehydrated in 100 mM NH4HCO3 with 1 pmol trypsin at 37 °C overnight. After digestion, the gel slices were washed with 0.1% trifluoroacetic acid (TFA) in 50% (v/v) acetonitrile three times to extract the peptides. The combined supernatants were condensed to 10 µL by vacuum centrifugation. Samples were desalted using a Zip Tip C 18 TM (Millipore, Bedford, MA) according to the manufacturer’s instructions and eluted with 2 µL of 50% acetonitrile/water containing 0.1% TFA for mass spectrometric analysis. Mass Spectrometry and Database Search. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was performed for the purified tryptic digests using a quadrupole timeof-flight mass spectrometer (Q-TOF-2; Micromass, Altrincham, U.K.) in the Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Chinese Academy of Sciences. The peptides were loaded using nanoelectrospray with gold-
Figure 2. Changes in soluble solids content and firmness in peach fruit treated with P. membranefaciens and SA. Bars represent standard errors of the mean.
coated borosilicate glass capillaries (Micromass). The applied spray voltage was 800-1000 V, with a sample cone working on 25-40 V. The MCP detector working voltage was 2250 V, and energy adjustable collision cell was filled with pure argon gas. MS/MS data were processed using MassLynx 3.5 (Micromass) and searched against NCBInr protein sequence databases with the MS/MS ion searching program MASCOT (http:// www.matrixscience.com). Variable modifications selected for searching include carbamidomethylation of cysteine, oxidation of methionine, and N-terminal pyroglutamine. Viridiplantae was chosen for the taxonomic category, and 0.8 Da was used as the mass error tolerance. All MS/MS spectra for a sample were sequenced de novo using the software PeaksStudio 3.0 (Bioinformatics Solutions, Journal of Proteome Research • Vol. 6, No. 5, 2007 1679
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Figure 3. Comparison of proteome patterns of peach fruit in response to P. membranefaciens and SA. The spots with altered abundance are marked with arrows. Proteins were extracted from peach fruit 1 day after antagonist yeast or SA treatments according to the method described by Shen et al.27 First dimension was carried out using IEF, and the second dimension was on 15% SDS-PAGE. Gels were detected by CBB-staining. The numbers indicate differentially expressed proteins and correspond to the numbers indicated in Tables 2 and 3. (A and C) Proteome patterns of the control peach fruit; (B) proteome patterns of yeast-treated peach fruit; and (D) proteome patterns of SA-treated peach fruit.
Inc., Canada), and the top five candidate sequences for each MS/MS were combined into a single text-format search string. The search string was then submitted to MS-BLAST sequence similarity searching as described by Shevchenko et al.31 via the MS-BLAST Web interface http://dove.embl-heidelberg.de/ Blast2/msblast.html. Extraction and Assay for Antioxidant Enzymes’ Activity. At various time intervals after treatment (1, 2, 4, 6, and 8 day), samples from 10 fruit flesh in the second group without pathogen inoculation were obtained for the enzyme assay. A flesh sample of 5 g was collected in each treatment using a sampler (5 mm deep and 7 mm in diameter) and homogenized in 25 mL of 50 mM ice-cold sodium phosphate buffer (pH 7.8 for CAT, pH 7.0 for superoxide dismutase (SOD) and glutathione peroxidase (GPX), and pH 6.4 for PPO extraction) and 0.5 g of polyvinyl polypyrrolidone with a Kinematica tissue grinder (Crl-6010, Kriens-LU, Switzerland). The homogenate was centrifuged at 6730g for 40 min at 4 °C, and the resulting supernatants were used directly for enzyme assay. There were three replicates in each treatment for enzyme assay, and the experiment was conducted twice. CAT activity was determined by adding 0.2 mL of enzyme preparation to 2.8 mL of 40 mM hydrogen peroxide (dissolved with 50 mM sodium phosphate buffer, pH 7.0) as a substrate.16 The decomposition of H2O2 was measured by the decline in 1680
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absorbance at 240 nm with a UV-160 spectrophotometer (Shimadzu, Japan). The specific activity was expressed in units per milligram of protein, where one unit of catalase converts one µmol of H2O2 per minute. For SOD assay,16 the reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM nitroblue tetrazolium (NBT), 10 µM EDTA, 10 µM riboflavin, and 0.1 mL of enzyme extract. The mixtures were illuminated with a fluorescent lamp (60 µmol m-2 s-1) for 10 min and then the absorbance was determined at 560 nm. Identical solutions held in the dark served as blanks. One unit of SOD was defined as the amount of enzyme that caused a 50% decrease of the SOD-inhibitable NBT reduction. The specific activity was expressed as units per milligram of protein. GPX activity was assayed by the oxidation of NADPH at 340 nm, as described by Pagila and Valentine.32 The reaction mixture consisted of 50 mM K-phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.25 unit glutathione reductase (EC 1.6.4.2; Sigma-Aldrich), 10 mM glutathione, 0.20 mM NADPH, and 1 mM sodium azide. The reaction was initiated by addition of 1 mM H2O2. One unit will catalyze the oxidation by H2O2 of 1.0 µmol of reduced glutathione to oxidized glutathione per minute at pH 7.0 at 25 °C. PPO activity was determined by adding 1 mL of enzyme preparation to 2 mL of 0.5 mM catechol as a substrate
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Peach Fruit Proteome Table 2. Proteins Identified in Peach Fruit by Quadrupole Time-of-Flight Tandem Mass Spectrometera spot no.b
v C0 vC4 v C5 v C9 v C10 C12 V C13 V v C14 C15 V C16 V v C17 v C18 v C19 v C20 v C21 v C22 v C23 C24 V v C29
homologous protein
glutathione peroxidase polyphenol oxidase precursor catalase methionine sulfoxide reductase putative actin-depolymerizing factor 14-3-3-like protein B UDP-N-acetylglucosamine pyrophosphorylase-like hsp 70-like protein UDP-glucose dehydrogenase gamma-aminobutyrate transaminase subunit precursor isozyme 3 GTP-binding nuclear protein RAN-A1 Superoxide dismutase [Mn], mitochondrial precursor cherry-allergen PRUA1 auxin-induced protein cytosolic glutamine synthetase; GS cytosolic phospho-glucomutase actin 8 putative succinyl-CoA ligase beta-chain, mitochondrial precursor NAD-dependent isocitratedehydrogenase alpha subunit
Mr (kDa)/ pI
Mowse scorec/ threshold
queries matched
sequence coverage (%)
19.3/4.86 67.1/6.39 57.0/6.95 21.5/6.3 16.3/5.12 29.3/4.78 67.5/6.4
139/46 71/44 143/45 100/45 67/44 187/45 105/45
3 2 5 3 1 4 1
18 4 10 10 4 11 2
76.5/5.07 52.9/6.06 57.2/6.72
203/45 204/46 288/45
4 4 3
6 8 7
25.0/6.38 25.8/7.10
170/45 270/45
5 3
17.6/5.87 34.2/5.35 38.8/5.46 63.3/5.46 41.9/5.37 45.1/5.98
356/44 141/45 76/45 162/45 70/46 213/45
36.2/6.08
243/46
accession number
ratiod
Medicago truncatula Prunus armeniaca Prunus persica Fragaria xananassa Arabidopsis thaliana Nicotiana tabacum Oryza sativa
gi|92894222 gi|3282505 gi|32526568 gi|11342533 gi|7267407 gi|1848208 gi|53792734
2.18 6.49 3.39 4.77 2.40 -3.51 -2.26
Arabidopsis thaliana Colocasia esculenta Lycopersicon esculentum
gi|7269278 gi|29028306 gi|29837286
2.14 -2.15 -2.58
22 21
Nicotiana tabacum Hevealiensis
gi|1172835 gi|464775
3.71 3.80
5 2 1 4 2 4
43 4 4 8 5 10
Prunus avium Vigna radiata Glycine max Pisum sativum Arabidopsis thaliana Oryza sativa
gi|1513216 gi|1184121 gi|256143 gi|6272281 gi|1669389 gi|50910635
2.19 2.53 3.16 7.59 3.41 -3.13
3
14
Brassica napus
gi|28974496
2.80
organism
a Fruit were treated with P. membranefaciens for 10 min and then stored at 25 °C for 1 day. b v: showed that spot intensity g 2-fold increased, and V: showed that spot intensity g 2-fold decreased in yeast-treated fruit in comparison to control fruit. c Protein MASCOT scores greater than the thresholds are significant (p < 0.05). d Ratio, amount of target protein in control versus samples treated by antagonist yeast.
Table 3. Proteins Identified in Peach Fruit by Quadrupole Time-of-Flight Tandem Mass Spectrometera spot no.b
v C0 v C1 v C2 C4 V v C5 v C7 v C8 v C9 v C10 v C17 C25 V C27 V v C29
homologous protein
glutathione peroxidase major cherry allergen Pru av 1.0202 peroxiredoxin polyphenol oxidase precursor catalase thymidine diphospho-glucose 4-6-dehydratase homologue triosephosphate isomerase methionine sulfoxide reductase putative actin-depolymerizing factor GTP-binding nuclear protein RAN-A1 V-ATPase catalytic subunit A NADP-dependent malic enzyme (NADP-ME) NAD-dependent isocitratedehydrogenase alpha subunit
Mr (kDa)/ pI
Mowse scorec/ threshold
queries matched
sequence coverage (%)
organism
accession number
ratiod
19.3/4.86 17.3/4.98 28.6/5.17 67.1/6.39 57.0/6.95 30.0/6.27
139/46 221/45 114/45 71/44 143/45 359/44
3 4 2 2 5 6
18 32 11 4 10 27
Medicago truncatula Prunus avium Phaseolus vulgaris Prunus armeniaca Prunus persica Prunus armeniaca
Gi|92894222 Gi|44409474 gi|11558244 gi|3282505 gi|32526568 gi|2351580
2.44 2.56 2.82 -3.96 2.08 2.55
27.1/5.54 21.5/6.3 16.3/5.12 25.0/6.38 68.6/5.30 65.2/6.09 36.2/6.08
244/44 100/45 67/44 170/45 685/45 183/45 243/46
6 3 1 5 10 5 3
30 10 4 22 20 9 14
Coptis japonica Fragaria xananassa Arabidopsis thaliana Nicotiana tabacum Prunus persica Vitis vinifera Brassica napus
gi|556171 gi|11342533 gi|7267407 gi|1172835 gi|15982954 gi|1708924 gi|28974496
2.19 3.30 4.02 2.14 -2.66 -2.37 4.72
a Fruit were treated with SA for 10 min and then stored at 25 °C for 1 day. b v: showed that spot intensity g 2-fold increased, and V: showed that spot intensity g 2-fold decreased in yeast-treated fruit in comparison to control fruit. c Protein MASCOT scores greater than the thresholds are significant (p < 0.05). d Ratio, amount of target protein in control versus samples treated by antagonist yeast.
according to Qin et al.,10 and the change in absorbance at 398 nm (A398) was measured immediately. The activity was expressed as A398 per minute per milligram of protein. Isolation of mRNA and Northern Blot Analysis. RNA was isolated by the hot-phenol isolation protocol according to the method described by Shirzadegan et al. with minor modifications.33 Briefly, 8 g of flesh from 10 fruits was ground in liquid nitrogen and transferred to a tube containing 20 mL of prewarmed (65 °C) buffer with 7 mL of phenol and 13 mL of extraction buffer (50 mM, pH 9.0, Tris-HCl, 100 mM NaCl, and 1% SDS). The tube was votexed for 10 min to thoroughly mix the materials and phenol before 8 mL of chloroform was added. After a final 30 s vortexing, the tube was incubated for 10 min and centrifuged (5000g) for 15 min at room temperature. The supernatant was then transferred to a fresh tube, and total RNA was re-extracted with an equal volume of chloroform, followed by centrifugation at 5000g for 10 min at room temperature. The
final supernatant was transferred to a fresh tube, and one-third volume of 8 M LiCl2 was added. The RNA was allowed to precipitate overnight at 4 °C, and centrifuged at 12 000g for 20 min at 4 °C. The pellet was washed with 1 mL of 75% ethanol, centrifuged at 12 000g for 10 min at 4 °C, and allowed to airdry. Total RNA was resuspended in DEPC H2O, quantified, and stored at -80 °C. For cDNA synthesis, forward (5′-TACAGGCACATGGAAGGCT-3′) and reverse (5′-GAATCGCTCTTGCCTGTCT-3′) primers for CAT were used according to a published sequence.34 Aliquots of 10 µg of poly(A) RNA per lane were loaded in a 1.2% denaturing formaldehyde agarose gel, and hybridization was carried out as described by Sambrook et al.35 Statistics. All data were analyzed as a one-variable general linear model procedure (analysis of variance) with SPSS (SPSS, Inc., Chicago, IL). Mean separations were performed using the least significant difference test. Differences at P < 0.05 were Journal of Proteome Research • Vol. 6, No. 5, 2007 1681
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Table 4. Identification of Differentially Expressed Proteins from Peach Fruit with MASCOT and MS-BLASTa spot no.
homologous protein by MASCOT
top hit by MS/MS-MS BLAST
C0 C1
glutathione peroxidase major cherry allergen Pru av 1.0202
C2 C4 C5 C7
peroxiredoxin polyphenol oxidase precursor catalase thymidine diphospho-glucose 4-6-dehydratase homologue triosephosphate isomerase
Glutathione peroxidase 1 Major cherry allergen Pru av 1.0201 Peroxiredoxin precursor Polyphenol oxidase precursor Catalase isozyme 2 Thymidine diphospho-glucose 4-6-dehydratase homologue Triosephosphate isomerase, cytosolic Peptide methionine sulfoxide reductase Putative actin-depolymerizing factor 1 14-3-3-like protein C UDP-N-acetylglucosamine pyrophosphorylase-1 Heat shock 70 kDa protein Putative UDP-glucose dehydrogenase Gamma-aminobutyrate transaminase subunit isozyme 3
C8 C9
methionine sulfoxide reductase
C10
putative actin-depolymerizing factor
C12 C13
14-3-3-like protein B UDP-N-acetylglucosamine pyrophosphorylase-like hsp 70-like protein UDP-glucose dehydrogenase
C14 C15 C16
C19 C20 C21
gamma-aminobutyrate transaminase subunit precursor isozyme 3 GTP-binding nuclear protein RAN-A1 Superoxide dismutase [Mn], mitochondrial precursor cherry-allergen PRUA1 auxin-induced protein cytosolic glutamine synthetase; GS
C22
cytosolic phospho-glucomutase
C23 C24
actin 8 putative succinyl-CoA ligase beta chain, mitochondrial precursor V-ATPase catalytic subunit A
C17 C18
C25 C27 C29
NADP-dependent malic enzyme NAD-dependent isocitrate dehydrogenase alpha subun
GTP-binding nuclear protein RAN-A1 Superoxide dismutase [Mn], mitochondrial precursor Major allergen Pru av 1 Auxin-induced protein Cytosolic glutamine synthetase GSbeta1 Phosphoglucomutase, cytoplasmic Actin Succinyl-CoA ligase [GDP-forming] beta-chain Vacuolar H+-ATPase A2 subunit isoform Malate oxidoreductase Putative (NAD+) isocitrate dehydrogenase
de novo sequence
HSPs scoreb
reference organism
CGLTNSNYTELNELY SDESTSVIPPPRIF
78 102
Helianthus annuus Prunus avium
GLGDLNYPLLSDVTK FDVFINDDAESLSR SIWISYWSQADK VVSNFIAQAIRDDPLTVQAPGTQTR
101 77 99 173
Phaseolus vulgaris Prunus armeniaca Gossypium hirsutum Prunus armeniaca
YGGSVDGANSK SGLYYYDETEVGYSE IFFIAWSPDTSR
70 102 96
Coptis japonica Fragaria x ananassa Oryza sativa
LAEQAERYEEMVEFMEK VLNVEYNQLDPLLR
136 100
Tropaeolum majus Oryza sativa
AVVTVPAYFNDSQRQA LAANAFLAQRLSLYDPQVSE
110 132
Mesembryanthemum crystallinum Oryza sativa
FTDSGSQADDTQVK
82
Lycopersicon esculentum
LPNQQTVDYPSFK
99
Arabidopsis thaliana
EVKKLVVETTANQDPLVTKGPTLVPLLG IDVWEHAYYLQYK TYESEFTSELPPPR EKYEELLLKFDVVYDTVGESDR VLAEYLWLGGSGMDLR
275
Hevea brasiliensis
106 102 115
Prunus avium Vigna radiata Glycine max
PTSAALDVVAQHLNLK
82
Populus tremula
GYMFTTTAER VPVDVFTGLTDEDAAK
79 101
Prunus persica Arabidopsis thaliana
GYNVSMMADSTSR
100
Lycopersicon esculentum
VFVSTQANNAYIFPGFGLGLVISGAIR ANPTALLLSSVTMLR
193 85
Arabidopsis thaliana Arabidopsis thaliana
a The Search string was submitted to MS BLAST sequence similarity searching via the Web interface http://dove.embl-heidelberg.de/Blast2/msblast.html. MS BLAST match was defined as statistically significant if the score of the HSP was higher than the threshold value in the query. The details of statistical evaluation and scoring scheme are described by Shevchenko et al.31
b
considered to be significant. Results presented were pooled across repeated experiments.
Results Incidence and Severity of Blue Mold. Decayed peach fruit first showed watery spots, and then the typical symptom of blue mold developed gradually from the inoculation site (Figure 1A). The symptom appeared in the control fruit at 2 days, but in treated fruit at 3 days. Compared with the control, both treatments could reduce disease incidence at 4 days after treatments, and decrease lesion diameter at 8 days after treatments (Figure 1B). This indicated that P. membranefaciens and SA treatments enhanced the resistance of peach fruit and delayed the initiation infection by P. expansum. Antagonist P. membranefaciens and SA treatments could increase soluble solids content (SSC) in harvested peach fruit 2 and 4 days after treatments. Firmness of the fruit declined with increasing storage time, but there was no significant difference between treated and untreated fruit (Figure 2). 2-DE Analysis of Proteins Induced by P. membranefaciens and SA. After 2-DE, changes in spot intensity between treated and control fruit samples were quantified using the software ImageMaster 2D Platinum 5.0. Protein spots were scored only when they were reproducibly observed in three independent replicates. Compared to the control, abundances of 43 proteins were differently expressed in yeast-treated fruit after 24 h, in which 25 proteins were up-regulated and 18 proteins were 1682
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Figure 4. Comparative distribution of proteins induced by P. membranefaciens and SA. In total, 25 proteins were identified by Q-TOF. The Venn diagram showed that 7 proteins (28%) were regulated by both antagonist yeast and SA, including antioxidant and PR-proteins.
down-regulated. In SA-treated fruit, abundances of 18 and 13 proteins were increased and decreased, respectively. Identification of Differentially Expressed Proteins with MS/ MS. Using MS/MS, we identified 25 proteins demonstrating statistically significant changes (p ) 0.05) in relative protein abundances in treated peach fruit (Figure 3; Tables 2 and 3). These proteins were searched with the MS/MS ion searching
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Figure 5. Time-dependent accumulation of antioxidant proteins in peach fruit treated with P. membranefaciens or SA 1 day after treatments. The relative abundance ratio of protein (% Volume) was determined. The protein spots with significant changes in intensities (p ) 0.05) were considered to be different. The graph represents an average of three spots from different gels.
program MASCOT, and further assessed with MS-BLAST (Table 4). Among them, 7 proteins (spots C0, C4, C5, C9, C10, C17, and C29) were induced by both antagonist P. membranefaciens and SA treatments (Figure 4).
The identified proteins could be grouped into five functional classes according to Bevan et al.36 and Schiltz et al.37 Those proteins were involved in metabolism, energy pathway, defense resistance, transcription, and cell structure. Journal of Proteome Research • Vol. 6, No. 5, 2007 1683
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Figure 6. Time-dependent accumulation of PR-proteins in peach fruit treated with P. membranefaciens and SA 1 day after treatments. The relative abundance ratio of protein (% Volume) was determined. The protein spots with significant changes in intensities (p ) 0.05) were considered to be different. The graph represents an average of three spots from different gels.
First, it is noteworthy that the abundances of 6 antioxidant proteins were changed after yeast and SA treatment, and 4 of them were regulated by both treatments, including glutathione peroxidase (GPX) (spot C0), polyphenol oxidase (PPO) precursor (spot C4), catalase (spot C5), and methionine sulfoxide reductase (MSR) (spot C9). Other antioxidant proteins, peroxiredoxin (spot C2) and superoxide dismutase (SOD) [Mn] (spot C18), were induced by SA and yeast treatment, respectively. Second, pathogenesis-related (PR)-proteins, such as major cherry allergen Pru av 1.0202 (spot C1) (induced by SA), cherry-allergen PRUA1 (spot C19) (induced by yeast), and glutathione peroxidase (spot C0) (induced by both), were identified after treatments. In addition, some proteins were identified as enzymes involving in the pathway of tricarboxylic acid cycle phosphorylation, including triosephosphate isomerase (spot C8), UDP-glucose dehydrogenase (spot C15), cytosolic phosphoglucomutase (spot C22), putative succinyl-CoA ligase beta-chain (spot C24), NADP-dependent malic enzyme (spot C27), and NAD-dependent isocitrate dehydrogenase alpha subunit (spot C29). Among them, spots C8, C27, and C29 were regulated by SA, and spots C15, C22, C24, and C29 were regulated by P. membranefaciens. Changes in the Abundances of Antioxidant and PR Proteins. The abundances of antioxidant proteins in a timedependent manner were shown in Figure 5. Peach fruit showed 1684
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higher GPX (C0) abundance at 2 days after yeast and SA treatments. Only SA enhanced the abundance of peroxiredoxin (C2) at 2 days after treatment. Yeast treatment enhanced, but SA treatment decreased, PPO precursor (C4) abundance at 1 day after treatments. In addition, the abundance of CAT (C5) was up-regulated by the yeast at 2 days and SA at 1 day, but there was no obvious difference thereafter. Both yeast and SA could increase MSR (C9) abundance in 1day. For SOD, yeasttreated fruit showed higher protein abundances than those in the control group at 2 days. The relative abundances of PR proteins were shown in Figure 6. The levels of major cherry allergen Pru av 1.0202 (spot C1) and cherry-allergen PRUA1 (spot C19) were up-regulated by SA and the yeast, respectively, at the first 2 days after treatments, but no evident differences were observed thereafter. The abundance of GPX (PR-9 protein) was increased in both yeastand SA-treated peach fruit as described above. Changes of Antioxidant Enzyme Activities after Treatments. The changes in activities of antioxidant enzymes were shown in Figure 7. Activity of GPX in treated peach fruit showed higher levels than those in the untreated control fruit during the whole storage stage. In control fruit, CAT activity decreased gradually with prolonged storage time. Treatments with P. membranefaciens and SA significantly (p ) 0.05) enhanced CAT activity in peach fruit, and the performance of the yeast showed to be
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Figure 7. Effects of P. membranefaciens and SA on enzyme activity in peach fruit stored at 25 °C. Bars represent standard errors of the mean.
better than that of SA. For SOD activity, there were no significant differences between treated fruit and control fruit at 2 days after treatments (p ) 0.05). But SOD activity declined slowly in treated fruit at 4 and 6 days after treatments. PPO activities in yeast- and SA-treated fruit were lower than those in the control fruit during the storage periods. Characterizations of the Expression of CAT Gene by Northern Blot Analysis. Among 5 antioxidant proteins, only CAT gene sequence of peach fruit is available in NCBI database. On the basis of the result of Northern blot analysis, message RNA from all fruits showed clear transcript accumulation of CAT gene at 1 day, but no evident difference was observed between treated and control fruit. Then at 2 days after treatments, the expressions of CAT mRNAs in both treated fruits showed to be stronger than those in the control mRNAs, especially in SA-treated fruit, but there was no obvious difference at 4 and 6 days (Figure 8).
Discussion Salicylic acid, as a signaling molecule, was proven to stimulate defense response in several plants.38-40 Recently, SA has been found to enhance disease resistance and have a potential for control of postharvest decay in some fruits.41,42 P. membranefaciens isolated from the surface of apple fruit has been approved to be a beneficial biocontrol agent and can effectively reduce postharvest diseases in various fruits.11,12 In this study, application of P. membranefaciens and SA could significantly delay the initiation infection process of P. expansum in peach fruit (Figure 1). On the basis of proteomic analysis, some defense-related proteins induced by P. membranefaciens and SA were identified. The first class consists of 6 antioxidant proteins (Figure 5; Tables 2 and 3). Among them, glutathione peroxidase (spot C0), catalase (spot C5), and
Figure 8. CAT gene expression in peach fruit stored at 25 °C after treatment with P. membranefaciens and SA. Accumulation of CAT mRNA was analyzed by Northern blots. Ten micrograms of separated RNA was hybridized with 32P-labeled CAT cDNA. The ethidium bromide-stained gel is below as a loading control.
peroxiredoxin (spot C2) are important for scavenging the reactive oxygen species (ROS).33,43,44 In addition, the function of methionine sulfoxide reductase (spot C9) and polyphenol oxidase precursor (spot C4) arerelated to the repair of oxidative damage intermediates.45,46 All these proteins are considered the main enzymatic systems for protecting cells against oxidative damage and responsible for the disease resistance.47-50 Interestingly, 4 antioxidant proteins were modulated by both P. membranefaciens and SA (Figure 3; Tables 2 and 3). These results suggested that the induction of the yeast and SA was associated with ROS metabolism. The second class includes 3 PR proteins (Figure 6; Tables 2 and 3). A substantial body of evidence has demonstrated that SAR is associated with the production of PR proteins, but ISR does not involve expression of PR-proteins.6,7 However, our Journal of Proteome Research • Vol. 6, No. 5, 2007 1685
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Figure 9. Proteins, whose abundances were regulated by P. membranefaciens and SA, were identified as enzymes involved in energy pathway. Step 1, catalyzed by UDP-glucose dehydrogenase (C15); step 2, catalyzed by cytosolic phosphoglucomutase (C22); step 3, catalyzed by triosephosphate isomerase (C8); step 4, catalyzed by NAD-dependent isocitrate dehydrogenase (C29); step 5, catalyzed by putative succinyl-CoA ligase (C24); step 6, catalyzed by NADP-dependent malic enzyme (C27).
results showed that both the yeast and SA treatments increased the activities of PR-9 and PR-10 proteins in peach fruit. The abundances of cherry allergen PRU A1 (spot C19) and cherry allergen Pru av 1 (spot C1) were elevated by P. membranefaciens and SA, respectively. These proteins belong to the ubiquitous family of plant PR proteins (PR-10).51,52 Peroxidase (spot C0), another PR-protein (PR-9),53 was enhanced in peach fruit treated with P. membranefaciens and SA. These PR proteins take an important role in defense-response against various pathogens. Proteins belonging to the third class are related to posttranscriptional modification (Figure 3; Tables 2 and 3). Of them, GTP-binding protein (spot C17) was co-modulated by P. membranefaciens and SA, which is involved in nucleocytoplasmic transport and is required for the import of protein into the nucleus and also for RNA export.54 Other 3 proteins, including 14-3-3 protein (spot C12), and heat shock protein 70 (spot C14), were regulated by P. membranefaciens. These proteins have been proven to play diverse roles in many biological processes involving transcriptional regulation,55,56 molecular chaperone,57 and membrane traffic regulation,58 respectively. The results indicated that these proteins were involved in the defense response of peach fruit and might play a prominent role in the recognition and execution of foreign invaders. Additionally, some proteins were identified as enzymes involved in glycolysis and tricarboxylic acid cycle. In the present study, 6 enzymes, which catalyze the reactions during sugar metabolism and energy pathway, were found to be altered after treatments with antagonist yeast or SA as compared to the control. After SA treatment, activities of triosephosphate isomerase (C8) and NAD-dependent isocitrate dehydrogenase 1686
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alpha subunit (C29) were enhanced, and activity of NADPdependent malic enzyme (NADP-ME) (C27) was decreased. These three enzymes catalyze steps 3, 4, and 6, respectively (Figure 9). This indicated that sugar metabolism was activated by SA treatment. After antagonist yeast treatment, the metabolism pathway of peach fruit was changed in a more complex manner (Figure 9). Decreased activity of UDP-glucose dehydrogenase (C15) (catalyzes step 1) and increased activity of NAD-dependent isocitrate dehydrogenase alpha subunit (C29) (catalyzes step 4) may result in activition of glycolysis and tricarboxylic acid cycle. On the other hand, enhanced activities of cytosolic phosphoglucomutase (C22) (catalyzes step 2) and putative succinyl-CoA ligase beta-chain (C24) (catalyzes step 5) may lead to hindrance of the whole metabolism pathway. These observations suggest that glycolysis and tricarboxylic acid cycle may be induced by the treatments of antagonist yeast and SA. Recently, the hypothesis of the model of SA action has been the subject of intense debate. One possible model is that SA inhibits the hydrogen peroxide (H2O2)-degrading activity of catalase, thereby leading to an increase in the endogenous level of H2O2. The H2O2, and other reactive oxygen species derived from it, may then serve as second messengers to activate the expression of plant defense-related genes and strengthen mechanical barriers.59,60 However, our results in this study showed that SA has no such influence. Instead, exogenous SA application enhanced the activity of catalase (Figures 5 and 7) and CAT expression (Figure 8) in peach fruit. Ananieva et al. also reported that CAT activities in barley were increased after SA treatment.61 Hence, the role of SA to mediate plant defense by inhibiting catalase, and thereby increasing the concentration of H2O2, is now controversial.62-64
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Taken together, the 2-DE-based proteomic approach exploits a new route to ellucidate defense mechanisms. The novel findings of this study included: (i) antioxidant proteins and PR-proteins are co-mediated by antagonist P. membranefaciens and SA; (ii) both ISR and SAR are associated with the production of some PR proteins; and (iii) treatments with antagonsit yeast and SA may change the metabolisms of glycolysis and tricarboxylic acid cycle. Our data also revealed that exogenous SA has no effect on inhibiting CAT activity in harvested fruit, which led to the changes of endogenous level of H2O2. These findings offer a mechanistic framework for the pathway of induced resistance by antagonist yeast and SA. However, the nature of induced resistance cannot be addressed thoroughly here, nor can its functional significance. Further studies on the protein and sugar metabolisms will be necessary to illuminate the mechanisms of ISR and SAR.
Acknowledgment. We thank Dr. Shihua Shen for his advice in the proteomics experiment, Ms. Mei Huang for her help in analysis of MS/MS, and Dr. Li Li for her suggestions. This study was supported by the National Natural Science Foundation of China (30430480; 30225030) and the Chinese Academy of Sciences (KSCX2-YW-N-007). Supporting Information Available: The detailed information of Mascot searches including the information of the mass spectra matching, the total number of peptides identified for each protein, and the amino acid sequence of each peptide are given in Supplemental Data 1; the results of MS BLAST searches for proteins that were identified with MS/ MS spectra are provided as Supplemental Data 2, including top de novo candidate sequences, high scoring pairs, and HSPs scores. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ross, A. F. Plant Physiol. 1961, 97, 1342-1347. (2) Tyler, B. M. Annu. Rev. Phytopathol. 2002, 40, 137-167. (3) Mclntyre, J. L.; Dodds, J. A.; Hare, J. D. Phytopathology 1981, 71, 297-301. (4) Dong, H.; Delaney, T. P.; Bauer, D. W.; Beer, S. V. Plant J. 1999, 20, 207-215. (5) Ton, J.; Mauch-Mani, B. Plant J. 2004, 38, 119-130. (6) Hammerschmidt, R. Physiol. Mol. Plant Pathol. 1999, 55, 77-84. (7) Welling, L. L. Trends Plant Sci. 2001, 6, 445-447. (8) Durner, J.; Shah, J.; Klessig, D. F. Trends Plant Sci. 1997, 2, 266274. (9) van Loon, L. C. Eur. J. Plant Pathol. 1997, 103, 753-765. (10) Qin, G. Z.; Tian, S. P.; Xu, Y.; Wan, Y. K. Physiol. Mol. Pqlant Pathol. 2003, 62, 147-154. (11) Fan, Q.; Tian, S. P. Plant Dis. 2000, 84, 1212-1216. (12) Qin, G. Z.; Tian, S. P.; Xu, Y. Postharvest Biol. Technol. 2004, 31, 51-58. (13) Wilson, C. L.; Chalutz, E. Sci. Hortic. 1989, 40, 105-112. (14) Droby, S.; Vinokur, V.; Weiss, B.; Cohen, L.; Daus, A.; Goldschmidt, E. E.; Porat, R. Phytopathology 2002, 92, 393-399. (15) El-Ghaouth, A.; Wilson, C. L.; Wisniewski, M. Phytopathology 2003, 93, 344-348. (16) Wang, Y. S.; Tian, S. P.; Xu, Y.; Qin, G. Z.; Yao, H. J. Postharvest Biol. Technol. 2004, 34, 21-28. (17) Chan, Z. L.; Tian, S. P. Postharvest Biol. Technol. 2006, 39, 314320. (18) Bae, M. S.; Cho, E. J.; Choi, E. Y.; Park, O. K. Plant J. 2003, 36, 652-663. (19) Lee, S.; Lee, E. J.; Yang, E. J.; Lee, J. E.; Park, A. R.; Song, W. H.; Park, O. K. Plant Cell 2004, 16, 1378-1391. (20) Kim, S. T.; Kim, S. G.; Hwang, D. H.; Kang, S. Y.; Kim, H. J.; Lee, B. H.; Lee, J. J.; Kang, K. Y. Proteomics 2004, 4, 3569-3578. (21) Peck, S. C.; Nu ¨ hse, T. S.; Hess, D.; Iglesias, A.; Meins, F.; Boller, T. Plant Cell 2001, 13, 1467-1475. (22) Ventelon-Debout, M.; Delalande, F.; Brizard, J.; Diemer, H.; Dorsselaer, A. V.; Brugidou, C. Proteomics 2004, 4, 216-225.
research articles (23) Hjernø, K.; Alm, R.; Canba¨ck, B.; Matthiesen, R.; Trajkovski, K.; Bjo¨rk, L.; Leopstorff, P.; Emanuelsson, C. Proteomics 2006, 6, 1574-1587. (24) Barraclough, D.; Obenland, D.; Laing, W.; Carroll, T. Postharvest Biol. Technol. 2004, 32, 175-181. (25) Sarry, J. E.; Sommerer, N.; Sauvage, F. X.; Bergoin, A.; Rossignol, M.; Albagnac, G.; Romieu, C. Proteomics 2004, 4, 201-215. (26) Tian, S. P.; Xu, Y.; Jiang, A. L.; Gong, Q. Q. Postharvest Biol. Technol. 2002, 24, 335-340. (27) Shen, S. H.; Jing, Y. X.; Kuang, T. Y. Proteomics 2003, 3, 527-535. (28) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (29) Komatsu, S.; Muhammad, A.; Rakwal, R. Electrophoresis 1999, 20, 630-636. (30) Shen, S. H.; Sharma, A.; Komatsu, S. Biol. Pharm. Bull. 2003, 26, 129-136. (31) Shevchenko, A.; Sunyaev, S.; Loboda, A.; Shevchenko, A.; Bork, P.; Ens, W.; Standing, K. Anal. Chem. 2001, 73, 1917-1926. (32) Pagila, D. E.; Valentine, W. N. J. Lab. Clin. Med. 1967, 70, 158169. (33) Shirzadegan, M.; Christie, P.; Seemann, J. R. Nucleic Acids Res. 1991, 19, 6055. (34) Bagnoli, F.; Danti, S.; Magherini, V.; Cozza, R.; Innocenti, A. M.; Racchi, M. L. Funct. Plant Biol. 2004, 31, 349-357. (35) Sambrook, J.; Fritisch, E. F.; Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 2001. (36) Bevan, M.; Bancroft, I.; Bent, E, Love, K.; Goodman, H.; Dean, C.; Bergkamp, R.; Dirkse, W.; Van Staveren, M.; Stiekema, W.; Drost, L.; Ridley, P.; Hudson, S. A.; Patel, K.; Murphy, G.; Piffanelli, P.; Wedler, H.; Wedler, E.; Wambutt, R.; Weitzenegger, T.; Pohl, T. M.; Terryn, N.; Gielen, J.; Villarroel, R.; De Clerck, R.; Van Montagu, M.; Lecharny, A.; Auborg, S.; Gy, I.; Kreis, M.; Lao, N.; Kavanagh, T.; Hempel, S.; Kotter, P.; Entian, K. D.; Rieger, M.; Schaeffer, M.; Funk, B.; Mueller-Auer, S.; Silvey, M.; James, R.; Montfort, A.; Pons, A.; Puigdomenech, P.; Douka, A.; Voukelatou, E.; Milioni, D.; Hatzopoulos, P.; Piravandi, E.; Obermaier, B.; Hilbert, H.; Dusterhoft, A.; Moores, T.; Jones, J. D.; Eneva, T.; Palme, K.; Benes, V.; Rechman, S.; Ansorge, W.; Cooke, R.; Berger, C.; Delseny, M.; Voet, M.; Volckaert, G.; Mewes, H. W.; Klosterman, S.; Schueller, C.; Chalwatzis, N. Nature 1998, 391, 485-488. (37) Schiltz, S.; Gallardo, K.; Huart, M.; Negroni, L.; Sommerer, N.; Burstin, J. Plant Physiol. 2004, 135, 2241-2260. (38) Rao, M. V.; Paliyath, G.; Ormrod, D. P.; Murr, D. P.; Watkins, C. B. Plant Physiol. 1997, 115, 137-149. (39) Tenhaken, R.; Ru ¨ bel, C. Plant Physiol. 1997, 115, 291-298. (40) Thulke, O.; Conrath, U. Plant J. 1998, 14, 35-42. (41) Yao, H. J.; Tian, S. P. Postharvest Biol. Technol. 2005, 35, 253262. (42) Zeng, K. F.; Cao, J. K.; Jiang, W. B. J. Sci. Food Agric. 2006, 86, 694-698. (43) Rouhier, N.; Gelhaye, E.; Sautiere, P. E.; Brun, A.; Laurent, P.; Tagu, D.; Gerard, J.; de Fay, E.; Meyer, Y.; Jacquot, J. P. Plant Physiol. 2001, 127, 1299-1309. (44) Vergauwen, B.; Pauwels, F.; Jacquemotte, F.; Meyer, T. E.; Cusanovich, M. A.; Bartsch, R. G.; van Beeumen, J. J. J. Biol. Chem. 2001, 276, 20890-20897. (45) Chevalier, T.; de Rigal, D.; Mbeguie-A-Mbeguie, D.; Gauillard, F.; Richard-Forget, F.; Fils-Lycaon, B. R. Plant Physiol. 1999, 119, 1261-1270. (46) Lowther, W. T.; Brot, N.; Weissbach, H.; Honek, J. F.; Matthews, B. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6463-6468. (47) Haraguchi, C. M.; Mabuchi, T.; Yokota, S. J. Histochem. Cytochem. 2003, 51, 215-226. (48) Herbette, S.; Le Menn, A.; Rousselle, P.; Ameglio, T.; Faltin, Z.; Branlard, G.; Eshdat, Y.; Julien, J. L.; Drevet, J. R.; Roeckel-Drevet, P. Biochim. Biophys. Acta 2005, 1724, 108-118. (49) Jain, M.; Brenner, D. A.; Cui, L.; Lim, C. C.; Wang, B.; Pimentel, D. R.; Koh, S.; Sawyer, D. B.; Leopold, J. A.; Handy, D. E.; Loscalzo, J.; Apstein, C. S.; Liao, R. Circ. Res. 2003, 93, e9-e16. (50) Taylor, A. B.; Benglis, D. M.; Dhandayuthapani, S.; Hart, P. J. J. Bacteriol. 2003, 185, 4119-4126. (51) Karlsson1, A. L.; Alm, R.; Ekstrand1, B.; Fjelkner-Modig, S.; Schio¨tt, Å.; Bengtsson, U.; Bjo¨rk, L.; Hjernø, K.; Roepstorff, P.; Emanuelsson, C. S. Allergy 2004, 59, 1277-1284. (52) Markovic´-Housley, Z.; Degano, M.; Lamba, D.; von RoepenackLahaye, E.; Clemens, S.; Susani, M.; Ferreira, F.; Scheiner, O.; Breiteneder, H. J. Mol. Biol. 2003, 325, 123-133. (53) van Loon, L. C.; van Strien, E. A. Physiol. Mol. Plant Pathol. 1999, 55, 85-97. (54) Merkle, T.; Haizel, T.; Matsumoto, T.; Harter, K.; Dallmann, G.; Nagy, F. Plant J. 1994, 6, 555-565.
Journal of Proteome Research • Vol. 6, No. 5, 2007 1687
research articles (55) de Ventten, N. C.; Ferl, R. J. Plant Physiol. 1992, 106, 1593-1604. (56) Ferl, R. J. Plant Mol. Biol. 1996, 47, 49-73. (57) Nelson, R. J.; Ziegelhoffer, T.; Nicolet, C.; Werner-Washburne, M.; Craig, E. A. Cell 1992, 71, 97-105. (58) Boman, A. L.; Kahn, R. A. Trends Biochem. Sci. 1995, 20, 147150. (59) Lamb, C.; Dixon, R. A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997, 48, 251-275. (60) de Gara, L.; de Pinto, M. C.; Tommasi, F. Plant Physiol. Biochem. 2003, 41, 863-870.
1688
Journal of Proteome Research • Vol. 6, No. 5, 2007
Chan et al. (61) Ananieva, E. A.; Christov, K. N.; Popova, L. P. J. Plant Physiol. 2003, 161, 319-328. (62) Bowler, C.; van Montagu, M.; Inze, D. Plant Mol. Biol. 1992, 43, 83-116. (63) van Wees, S. C. M.; Glazebrook, J. Plant J. 2003, 33, 733-742. (64) Dangl, J. L.; Dietrich, R. A.; Richberg, M. H. Plant Cell 1996, 8, 1793-1807.
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