Comparison of Two Proteomics Techniques Used to Identify Proteins

Dec 30, 2005 - Synopsis. Two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel elec...
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Comparison of Two Proteomics Techniques Used to Identify Proteins Regulated by Gibberellin in Rice Setsuko Komatsu,* Xin Zang, and Naoki Tanaka Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan Received September 1, 2005

Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. We compared two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), for their ability to identify proteins regulated by gibberellin (GA) in rice. Two-week-old rice seedlings were treated with or without 5 µM GA3 for 48 h and proteins extracted from the basal region of the leaf sheath. After separation of the proteins by the two techniques, the amino acid sequences of GA3-responsive proteins were analyzed using a protein sequencer and mass spectrometry. 2D-LC and 2D-DIGE were able to resolve 1248 protein fractions and 1500 proteins, respectively. Out of these, 2D-LC identified 9 proteins that were up-regulated and 9 that were down-regulated by GA treatment; 2D-DIGE identified 4 upregulated and 4 down-regulated proteins. The two techniques detected overlapping sets of proteins. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenase and photosystem II oxygen-evolving complex protein were identified as GA3-regulated proteins by both methods. In addition, these two methods uncovered GA3-regulated unknown proteins which had not been reported previously, and novel proteins which are not detected in 2D-PAGE followed by Coomassie brilliant blue staining. These results suggest that these two methods are among some of the very useful tools for detecting proteins that may function in various physiological and developmental processes in plants. Keywords: rice • leaf sheath • gibberellin • 2D-LC • 2D-DIGE

Introduction In the post-genomic era, proteomics is becoming increasingly important because proteins are directly related to function.1 Because proteins are involved in most processes in living cells, a detailed understanding of proteins is critical to the study of cells and organisms at the molecular level. Furthermore, nucleotide sequences provide only limited information about the protein complement encoded by the genome; posttranscriptional regulation often results in a lack of correlation between transcript levels and protein abundance.1 Proteomics must address questions ranging from the straightforward identification of proteins to the characterization of posttranslational modifications, protein-protein interactions, and subcellular localization. Hence, proteomics is becoming increasingly important for the investigation of complex cellular processes. It has also been used successfully for genetic and physiological studies.1 Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. With the availability of the genome sequences of some plants, plant proteomics is playing an increasingly important role in genome annotation and has recently been * To whom correspondence should be addressed. Tel: 81-29-838-7446. Fax: 81-298-38-7464. E-mail: [email protected].

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applied toward the understanding of plant development, disease resistance, photosynthesis, and other aspects of plant function.2-4 The past decade has seen considerable progress in the field of rice proteomics.5 Rice is one of the most important crops in the world, being the main staple food for more than half of the world’s population.6 Rice is also an excellent model plant for monocot crop species, because of its relatively small genome of about 440 Mb.7 The complete map-based genome sequences of the Nipponbare cultivar have been finished, and it is expected that the complete genome of japonica will be decoded and made available to the public.6 As part of the analysis of diverse complete genomes, gene prediction programs have been used to establish categories of proteins; coupled with studies of protein functions, these categories provide a useful classification scheme for genes and genomic sequences from additional organisms. To date, several proteomics studies of rice have revealed diverse functional categories of proteins. The Rice Proteome Database website (http://gene64.dna.affrc.go.jp/RPD/main.html) has been constructed and provides extensive information on the progress of rice proteome research.8 In previous studies of the rice proteome, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) coupled with direct protein sequencing and/or mass spectrometry (MS) was 10.1021/pr0502929 CCC: $33.50

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Rice Proteome Using Two Techniques

used for protein separation and identification. Ever since O’Farrell9 demonstrated that proteins could be resolved based on their isoelectric points and molecular weights by electrophoresis on polyacrylamide gels, 2D-PAGE has remained unchallenged as the most efficient way of analyzing complex protein mixtures. Modern large-format gels (e.g., 20 × 20 cm) are highly reproducible and Coomassie brilliant blue (CBB) or silver staining allows visualization of several hundred proteins.3 Color density and size of the detected spots enable protein quantification. The accuracy of these methods, however, is limited by the low dynamic range of most staining techniques. The recent development of fluorescent dyes for proteins12 overcomes this limitation. Despite the power of 2D-PAGE as a proteomics tool, the range of pI’s and molecular weights that can be resolved by this technique is limited. Because it is difficult to separate basic proteins (pH > 8.0) using isoelectric focusing (IEF) gels prepared according to the method of O’Farrell,9 immobilized pH gradient gels, which cover pI’s ranging from 6.0 to 10.0, have also been used in the first dimension.13 These limitations of protein separation must be overcome to enable identification of all the proteins in a given proteome. 2D liquid chromatography (LC), which combines pH gradient and reversed-phase columns, is a new proteomics technique that promises to extend the range of protein separation. The isotope coded affinity tag (ICAT) technique has recently been developed for quantitative comparisons in the absence of 2D-PAGE.10 However, problems relating to reproducibility and the number of replicates required for establishing statistical significance have yet to be completely resolved.11 Alternative “gel-less” approaches, such as multidimensional protein identification technology (MudPIT), have already been used effectively to catalog many polypeptides in total protein mixtures from several organisms, including rice.14,15 However, while MudPIT is an excellent means to generate an exhaustive catalog of proteins present in a particular protein sample, it does not yield reproducible quantitative information.16 Plant cell division, growth, and differentiation need to be precisely controlled during development to ensure the coordinated growth of tissues. Plant hormones are among the most important factors affecting these phenomena. Thus, the plant hormone gibberellin (GA) plays an essential role in many aspects of plant growth and development, such as seed germination, stem elongation, leaf expansion, and flower and fruit development.17 A close interaction between GA metabolism and GA response pathways has recently been revealed.18 The metabolism of GA and its response pathways also integrate with other signaling pathways to regulate plant growth, and, therefore, the effects of GA on development are likely to be extremely complex. In this study, two proteomics techniques, 2D-LC and fluorescence 2D difference gel electrophoresis (2DDIGE), were compared for their ability to identify proteins regulated by the plant hormone GA in rice.

Experimental Procedures Plant Material and Treatment. Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown in plastic seedling pots under white fluorescent light (600 µmol/m2/s, 12 h light period/ day) at 28 °C and 75% relative humidity in a growth chamber (Sanyo, Osaka, Japan). At 2 weeks after sowing, seedling pots were transferred to plastic containers containing 5 µM GA3 (Wako Pure Chemical, Osaka, Japan) for 48 h. The experiment was repeated 10 times; 5 times for the 2D-LC experiment and

research articles another 5 times for 2D-DIGE. Sixty seedlings, 30 control seedlings and 30 treatment seedlings, were used for each experiment. Two-Dimensional Liquid Chromatography Analysis. Basal regions of leaf sheaths (500 mg) were homogenized with 1 mL of 50 mM Tris-HCl (pH 8.0), and mixed with 1.2 mL of lysis buffer containing 7.5 M urea, 2.5 M thiourea, 12.5% glycerol, 50 mM Tris-HCl (pH 8.0), 2.5% n-octylglucoside, 6.25 mM Tris(carboxyethyl) phosphine hydrochloride and 1.25 mM protease inhibitor (Sigma, St. Louis, MO), using a glass mortar and pestle on ice. Each homogenate was centrifuged at 15 000 × g for 5 min and the supernatant removed. The pellet was washed with an additional 1.2 mL lysis buffer and recentrifuged. The combined supernatants were subjected to 2D-LC analysis using the ProteomeLab PF 2D kit (Beckman Coulter, Fullerton, CA) according to the manufacturer’s recommendation. For the first dimension, the supernatants (2 mg of protein) were injected onto a chromatofocusing column operated by AKTA explorer (Amersham Biosciences, Piscataway, NJ), and separated along pI gradients. For the second dimension, the pI fractions were separated using a reversed phase column operated by an HPLC system (Gilson, Lewis Center, OH). The HPLC data were analyzed to compare control and treatment, and were visualized as a ladder pattern with ProteoVue software (Beckman Coulter). The experiment was repeated 5 times. Fluorescent Dye Labeling. Basal regions of leaf sheaths (500 mg) were homogenized with 1 mL of a lysis buffer9 containing 8 M urea, 2% Nonidet P-40 (NP-40), 0.8% Ampholine (pH 3.510 and pH 5-8, Amersham Biosciences), 5% 2-mercaptoethanol, and 5% poly(vinylpyrrolidone)-40, using a glass mortar and pestle on ice. Each homogenate was centrifuged at 15 000 × g for 5 min and the supernatant removed. The pellet was washed with an additional 1.2 mL lysis buffer and recentrifuged. The combined supernatants were mixed with 50% trichloroacetic acid to a final concentration of 10%. The solution was kept for 30 min on ice and centrifuged at 15 000 × g for 10 min. The resultant precipitates were washed with 100 µL ice-cold ethanol, suspended in labeling buffer and labeled with Cy5 or Cy3 fluorescent dye (Amersham Biosciences). Equal volumes of each fluorescent dye-labeled sample were mixed, added to lysis buffer, separated by 2D-PAGE as described below, and scanned using a Typhoon 8600k variable imager (Amersham Biosciences). The image map was analyzed with DeCyder software (Amersham Biosciences). The experiment was repeated 5 times. Two-Dimensional Polyacrylamide Gel Electrophoresis. For 2D-PAGE,9 samples were separated in the first dimension by IEF. IEF tube gels 11 cm in length and 0.3 cm in diameter were prepared with an IEF gel solution consisting of 8 M urea, 3.5% polyacrylamide, 2% Nonidet P-40, and 2% Ampholine (pH 3.510 and pH 5-8). Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 600 V for 1 h. After IEF, SDS-PAGE in the second dimension was performed using 15% polyacrylamide gels with 5% stacking gels. To analyze amino acid sequences, gels were stained with CBB or deep purple (Amersham Biosciences). Cleveland Peptide Mapping. Following protein separation by 2D-PAGE, gel pieces containing protein spots were excised and the proteins were electroeluted using an electrophoretic concentrator (Nippon-Eido, Tokyo, Japan) at 2W constant power for 2 h. After electroelution, the protein solution was dialyzed against deionized water for 2 days and lyophilized. Proteins were redissolved in 20 µL of SDS sample buffer containing 0.5 M Tris-HCl (pH 6.8), 10% glycerol, 2.5% SDS, Journal of Proteome Research • Vol. 5, No. 2, 2006 271

research articles and 5% 2-mercaptoethanol, and loaded onto an SDS-PAGE gel. The sample was overlaid with 20 µL of a solution containing 10 µL Staphylococcus aureus V8 protease (0.1 µg/µL; Pierce, Rockford, IL) and 10 µL of SDS sample buffer. Electrophoresis was performed until the sample and protease were stacked in the stacking gel, and then interrupted for 30 min to allow the protein to digest.19 N-Terminal and Internal Amino Acid Sequence Analysis. After 2D-PAGE and Cleveland peptide mapping, proteins and peptides, respectively, were transferred by electroblotting onto a poly(vinylidene difluoride) (PVDF) membrane (Pall, Port Washington, NY) and detected by CBB staining. Stained protein spots or peptide bands were excised from the PVDF membrane and directly subjected to Edman degradation in a gas-phase protein sequencer (Procise cLC, Applied Biosystems, Foster City, CA). The amino acid sequences determined by the protein sequencer were compared with protein sequences in the SwissProt database using the FASTA sequence alignment program. Mass Spectrometry Analysis. Proteins stained with CBB or deep purple after 2D-PAGE were prepared for MS from 3 to 5 gels or fractions as described previously.20 These proteins and proteins fractionated by 2D-LC were digested in 50 µL of 10 mM Tris-HCl (pH 8.0) containing 1 pM trypsin (Sigma) at 37 °C for 10 h. Purification of the resulting peptides was achieved using Zip-Tips (Millipore, Bedford, MA). The purified peptides (2 µL) were added directly to a 10 mg/mL R-cyano-4-hydroxycinnamic acid, 0.3% trifluoroacetic acid, and 50% acetonitrile matrix, and air-dried onto a plate for analysis using matrixassisted laser desorption-ionization time-of flight MS (MALDITOF MS, Voyager-DE RP, Applied Biosystems). Matching of empirical peptide mass values with theoretical digests and sequence information obtained from the database was performed using Mascot software (Matrix Science Ltd., London, UK). The experiments were done 2 to 4 times. For MALDI-TOF MS analysis, 4 criteria were used to assign a positive match with a known protein: (I) the deviation between the experimental and theoretical peptide masses needed to be less than 50 ppm; (II) at least 6 different predicted peptide masses needed to match the observed masses for an identification to be considered valid; (III) the matching peptides needed to cover at least 30% of the known protein sequence; and (IV) Individual ions scores >51 identity or extensive homology (P < 0.05). Northern Blot Analysis. Basal part of leaf sheath samples were quick-frozen in liquid nitrogen and ground to powder by mortar and pestle. Total RNAs were isolated according to the procedure of Chomcyznki and Sacchi.21 For Northern blot analysis, 20 µg of total RNA was separated on 1.2% agarose containing 6% formaldehyde and transferred onto a HybondN+ nylon membrane (Amersham Biosciences). Loading of equal amounts of total RNA for Northern blots was determined by visualization of ethidium bromide-stained rRNA bands. PCR products for probes were purified from agarose gels (QIAEXII, Gel Extraction Kit, Qiagen Sciences, Germantown, MD), and radio labeled using [R-32P] dCTP (Amersham Biosciences) random prime labeling system (Rediprime II, Amersham Biosciences). Hybridization was performed in the ultrasensitive hybridization buffer (ULTRAhyb) at 42 °C overnight. The blots were washed twice in 2X SSC, 0.1%X SDS at 42 °C for 10 min each and then twice in 0.1X SSC, 0.1% SDS at 68 °C for 15 min, and then analyzed by the phosphorimage program with the Typhoon 8600k variable imager (Amersham Biosciences). 272

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Results and Discussion Analysis of Gibberellin Effects on the Basal Part of the Leaf Sheath of Rice Seedlings. GA plays an essential role in many aspects of plant growth and development, such as seed germination, leaf sheath elongation, leaf expansion, and flower and fruit development.17 It is well established that GAs are responsible for triggering stem or internodal elongation. In rice seedlings, the basal part of the leaf sheath contains root primodia and meristem cells, which undergo active cell division and development.22 This functionally important region is the site of many critical metabolic and regulatory activities that eventually control the height and robustness of the plant. In previous studies, rice leaf sheath proteins were analyzed using 2D-PAGE with MS and/or direct protein sequencing and 783 proteins were identified).20,23,24 In the resulting proteome, proteins involved in central metabolism were the most abundant and no clear function could be predicted for 20% of the proteins.20 In this study, 2D-LC and 2D-PAGE with fluorescent dye labeling were used to identify proteins regulated during GA3stimulated elongation of leaf sheaths. To assess the effective conditions for promotion of rice leaf sheath elongation by GA3, excised leaf sheath segments of two-week-seedlings were treated with exogenous GA3 for different time periods.23 Exogenous application of GA3 in amounts as low as 0.1 µM promoted leaf sheath elongation, and the effect was saturated at 5 µM after incubation for 48 h. To further examine the kinetics of GA3 action on leaf sheath elongation, a time course experiment was conducted by treating leaf sheath segments with 5 µM GA3 for up to 72 h. The leaf sheath showed significant elongation within 6 h and the response peaked within 48 h of GA3 treatment. The previous report showed that the elongation of the leaf sheath is strongly stimulated by 5 µM GA3 within 48 h and this parameter was used in subsequent experiments.23 Therefore, in this study, the proteins were extracted from the basal part of leaf sheaths from 2-week-old rice seedlings treated with or without 5 µM GA3 for 48 h. Identification of Gibberellin-Regulated Proteins in the Basal Part of Leaf Sheath by 2D-LC. Proteins extracted from 5 µM GA3-treated and untreated tissues were purified with a ProteomeLab PF 2D kit, and separated on pI gradient and reversed phase columns. After 2D-LC, the image of the 2D map was analyzed and visualized using ProteoVue software (Figure 1). The resulting 2D map showed 9 proteins that were upregulated and 9 proteins that were down-regulated by GA3 (Figure 1). The up-regulated proteins, designated LC1, LC3, LC4, LC11, LC14, LC15, LC16, LC17, and LC18, were identified as unknown protein, photosystem II oxygen-evolving protein, heat-shock protein beta-1 (hsp27), superoxide dismutase [Cu-Zn], ATPase alpha subunit, cytosolic glyceralaldehyde-3phosphate dehydrogenase (GAPDH), acyl-CoA-binding protein, and 2 ribosomal proteins (Table 1). The down-regulated proteins, designated LC2, LC5, LC6, LC7, LC8, LC9, LC10, LC12, and LC13, included photosystem II oxygen-evolving protein, 2 GAPDHs, phosphate-binding periplasmic ABC transporter, and 4 proteins that could not be determined (Table 1). Photosystem II oxygen-evolving protein and GAPDHs were detected as both up-regulated and down-regulated proteins. We assume that they represent different members of a multigene family, or perhaps different forms of the same protein. Identification of Gibberellin-Regulated Proteins in the Basal Part of Leaf Sheath by 2D-DIGE. Proteins extracted from 5 µM GA3-treated and untreated tissues were labeled with Cy5

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Table 1. Proteins Regulated by Gibberellin Treatment in Leaf Sheaths of Rice Seedlings Separated by 2D-LC no.a

LC1 LC2 LC3 LC4 LC5 LC6 LC7 LC8 LC9 LC10 LC11 LC12 LC13 LC14 LC15 LC16 LC17 LC18

homologous protein

ac no.b

scorec

ratio

unknown Nascent polypeptide associated complex alpha chain Photosystem II oxygen-evolving protein Mitochondrial F0 ATP synthase δ chain Photosystem II oxygen-evolving protein Heat-shock protein beta-1 (Hsp27) GAPDH GAPDH Phosphate-binding periplasmic ABC transporter NDd ND ND Superoxide dismutase [Cu-Zn] 1 ND ND ATPase alpha subunit (atps 3) GAPDH Unknown protein Acyl-CoA-binding protein Putative 40S ribosomal protein S13 Putative 40S ribosomal protein S15

gi:37532862 gi:34907258 gi:482311 gi:50946875 gi:3491448 gi:55628780 gi:29150193 gi:24415114 gi:17546248

184 151 363 114 123 92 64 114 105

+5.4

gi:134595 gi:37533324 gi:50940735 gi:34901084 gi:55775158 gi:50940531 gi:50919151

94 105 62 124 104 202 100

-6.5 +10.4 +4.0 -9.8 -8.5 -3.5 -8.3 -7.5 +4.4 -3.2 -3.0 +3.0 +3.0 +4.8 +3.5 +5.4

a The nos. refer to the spot numbers as given in Figure 1. b Accession number in NCBI database. c Probability of a true positive identification. d Could not be determined. Cytosolic glyceraldehyde-3-phosphate dehydrogenase: GAPDH.

Figure 1. Detection of GA3-responsive proteins in the basal region of the leaf sheath by 2D-LC. Two-week-old seedlings were treated with (+) or without (-) 5 µM GA3 for 48 h. Proteins were extracted from the basal part of the leaf sheath and separated with a 2D-LC system (chromatofocusing for the first dimension and reversed phase for the second dimension). The analyzed data were visualized with ProteoVue software. Arrows show the positions of proteins increased or decreased by GA3 treatment. The experiments were performed in triplicate. The pI range of each pair of lanes (+ or -GA3) is indicated at the bottom of the figure. The position of each protein fraction in the vertical dimension correlates with its retention time on the reversed phase column (B). Panel A shows data for two independent control samples (treated without GA3 for 48 h).

and Cy3, respectively. After 2D-PAGE, the image of the 2D map was analyzed and visualized using DeCyder software (Figure 2). Eight GA3-regulated proteins were detected with this

method: 6 major ones in the low contrast analysis (Figure 2A) and 2 minor ones in the high contrast analysis (Figure 2B). The 4 proteins that were up-regulated by GA3 treatment, shown as FD1, FD2, FD3, and FD4 in the 2D map, were catalase, GAPDH, calreticulin, and photosystem II oxygen-evolving protein (Table 2). On the other hand, 4 proteins, shown as FD5, FD6, FD7, and FD8, were decreased by GA3 treatment; these were ATP sulfurylase (atps 3), a resistance gene analogue, and 2 unknown proteins (Table 2). Function of the Gibberellin-Regulated Proteins in the Basal Part of Leaf Sheath. Among the proteins increased by GA3 was photosystem II oxygen-evolving protein (LC3 and FD4). This protein was detected by both the 2D-LC system and 2D-DIGE, as well as in our previous studies.20,23 The up-regulation of photosystem II oxygen-evolving protein in leaf sheath shows that photosynthetic activity in this organ was increased by GA3 treatment. Catalase was also detected as a GA3-upregulated protein in the 2D-PAGE analysis. This enzyme is involved in reactive oxygen species scavenging reactions and detoxifies H2O2.25 H2O2 is produced by superoxide dismutase, which converts superoxide (O2-) to H2O2 in defense against reactive oxygen species. We propose two possible explanations for the up regulation of catalase in GA3-treated leaf sheath. First, growth responses are correlated with cell wall-loosening reactions and the degradation of cell wall polysaccharides.26 Hydroxyl radicals (•OH), produced by the reaction of H2O2 with O2-, have been reported to increase during the cleavage of polysaccharides such as hyaluronate, chitosan and pullulan.26 Therefore, increased catalase may be needed to scavenge H2O2 generated by cell wall-loosening during basal region elongation. Second, reactive oxygen species are also generated during photosynthesis.25 Therefore, the increased photosynthesis in GA3-treated leaf sheath (see above) may also increase the need for catalase. GAPDH (LC15 and FD4) was identified as a GA3-upregulated protein by both 2D-LC and 2D-DIGE, although 2 GAPDHs (LC5 and LC6) were down-regulated. In our previous studies, GAPDH was found to be increased20 and phosphorylated27 in response to GA3 during leaf sheath elongation. On the other hand, 2 GAPDHs down-regulated by GA3 are needed further physiJournal of Proteome Research • Vol. 5, No. 2, 2006 273

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Table 2. Proteins Regulated by Gibberellin Treatment in Leaf Sheaths of Rice Seedlings Separated by 2D-DIGE no.a

MMb

pIb

IDc

amino acid sequencesd

FD1 FD2 FD3 FD4

59.2 56.3 50.6 20.5

7.1 6.0 5.0 6.1

MS MS Ed Ed

(N-blocked) (N-blocked) N-KVFFQEKF N-AYGEAANVFG

FD5 FD6

34.7 26.5

5.0 5.6

MS Ed

(N-blocked) N-AAVAGEE

FD7

42.7

7.9

Ed

FD8

24.7

5.1

MS

N-SVSSAQIEES I-SGFETATVAD I-NQLITVKPDT (N-blocked)

homologous protein

%e

ac no.

ratio

Catalase GAPDH Calreticulin Photosystem II oxygenevolving protein ATP sulfurylase Oryza sativa resistance gene analog unknown

114 123 100 100

O62839 AF010582 P30806 P81668

+1.56 +1.59 +1.73 +1.73

56 67

AJ223498 AF146275

-1.44 -1.43

100

AB110184

-2.04

81

AP005933

-1.45

unknown

a The nos. refer to the spot numbers as given in Figure 2. b Molecular mass (MM) and pI are from the gel in Figure 2. c Methods of protein identification: MS, mass spectrometry; Ed, Edman degradation. d N-terminal (N-) and internal (I-) amino acid sequences as determined by Edman degradation. e The values in parentheses indicate the score for MS and the homology for the identity protein sequences for Ed, respectively. Cytosolic glyceraldehyde-3-phosphate dehydrogenase: GAPDH.

ological experiment. GAPDH plays an important role in glycolysis and gluconeogenesis by reversibly catalyzing the oxidation and phosphorylation of D-glyceraldehyde-3-phosphate to D-1, 3- biphosphoglycerate. The enzyme is NAD+ specific and found in the cytoplasm of both photosynthetic and nonphotosynthetic tissues. GADPH is also a crucial component of glycogen metabolism. The phosphorylation of GADPH detected after GA3 treatment of rice leaf sheath could mean that the enzyme is involved in GA signal transduction. However, the abundance of GADPH was also increased by GA3 treatment in rice leaf sheath27 and rice suspension-cultured cells.20 Therefore, the increased labeling of GADPH may reflect GAstimulated accumulation of the protein rather than a GA effect on phosphorylation. In wheat, the activity of nonphosphorylating GADPH was reported to be regulated by phosphorylation, divalent cations, and 14-3-3 proteins.28 Therefore, phosphorylation of GADPH after GA3 treatment suggests that GA3 activates the metabolism of rice leaf sheath cells to allow for increased growth.

Figure 2. Detection of GA3-responsive proteins in the basal region of the leaf sheath by 2D-DIGE. Two-week-old seedlings were treated with or without 5 µM GA3 for 48 h, and then proteins extracted from the basal region of the leaf sheath. The extracted proteins from GA3-treated and untreated tissues were labeled with Cy5 and Cy3 fluorescent dye, respectively. Equal volumes of the two fluorescent dye-labeled samples were mixed, separated by 2D-PAGE, and scanned using the Typhoon 8600k variable imager. The data were analyzed with DeCyder software. GA3-responsive proteins of relatively high abundance were detected by low contrast imaging (A), and low abundance changed proteins were detected by high contrast imaging (B). Red or blue letters indicate proteins that were increased or decreased, respectively, by GA3 treatment. 274

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Superoxide dismutase [Cu-Zn] (LC11) was up-regulated in this study and in a previous study.20 Superoxide dismutase [Cu-Zn] is an essential component of the pathogen defense mechanism in most organisms.29 Calreticulin (FD3) was also up-regulated in this study and a previous study.24 Calreticulin is a Ca2+-binding protein of the endoplasmic reticulum and also functions as a molecular chaperone.30 In pathogen-induced elicitor signaling in tobacco, calreticulin was identified as one of the oligogalacturonide-modulated phosphoproteins.31 Abbasi et al.32 reported that the calcium-dependent protein kinase OsCDPK13 might be an important signaling component in the response of rice to GA and cold. The connection to cold stress responses could be indirect, and OsCDPK13 could simply be involved in GA signaling. If this is the case, then calreticulin and superoxide dismutase [Cu-Zn] may also be involved in GA signaling and only indirectly connected to other stress responses. As shown in Figure 3, the abundance of photosystem II oxygen-evolving protein and GADPH was increased by GA3 However, the expression level of these mRNA did not changed by GA3 (Figure 3). Furthermore, photosystem II oxygen-evolving protein and GADPH showed continuous increase after sowing (Figure 4). These results indicate that the fundamentally important proteins for leaf sheath elongation are detected by multiple approaches. At least two proteins detected in this

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Figure 3. Protein abundance and mRNA expression changes of photosystem II oxygen-evolving protein and GADPH by GA3 in the basal region of leaf sheath. Rice leaf sheath were treated without (-) or with (+) 5µM GA3 for 6 h. For silver staining, only the protein spots for photosystem II oxygen-evolving protein (A) and GADPH (B) detected on 2D-gels were selected to show the changes of proteins. The transcriptional changes were detected by Northern blot analysis.

of immobilized pH gradient gels. These results suggest that 2DLC and 2D-DIGE are powerful methods for detecting previously missed changes to the proteome. Using these two techniques, many more proteins were detected compared with 2D-PAGE followed by CBB staining. Using the earlier method, GADPH and photosystem II oxygenevolving complex protein were detected as GA3-regulated proteins. However, the two new methods tested here detected GA3-regulated unknown and novel proteins that were not reported previously and are not detected by 2D-PAGE followed by CBB staining. The minor GA-responsive proteins were detected only by 2DDIGE. One of the decreased proteins in the basal region following GA3 treatment was ATP sulfurylase (FD5). ATP sulfurylase, the first enzyme of the sulfate assimilation pathway, is localized primarily in plastids, but there is also a minor cytosolic form.33 Some aspects of plant sulfur metabolism, which includes the transport and cycling/degradation of sulfur compounds, are still unclear.33 Nevertheless, the decrease in ATP sulfurylase after GA3 application indicates that GA regulates the first step of sulfur metabolism in rice plants. Low molecular weight proteins were detected only by the 2D-LC system. One of these was acyl-CoA-binding protein (LC16). Cytosolic 10-kDa acyl-CoA-binding proteins are prevalent in eukaryotes and are highly conserved across species, suggesting that their physiologicalroles have been preserved through evolution. In plants, fatty acids synthesized in the chloroplasts are exported as acyl-CoA esters to the endoplasmic reticulum.34 The increase in acyl-CoA-binding protein following GA3 treatment indicates that GA regulates lipid metabolism. These results suggest that 2D-LC and 2D-DIGE are among some of the very useful tools for detecting regulated proteins of low molecular weight or low abundance.

Concluding Remarks Figure 4. Protein expression during development of the rice basal region. Rice seedlings were grown for 2, 4, 6, 8, and 10 weeks after sowing. Proteins were extracted from the basal region in seedlings, separated by 2D-PAGE, and staining by CBB. Following scanning, the gel patterns were analyzed using the 2D Elite software, and relative abundance ratio of proteins for photosystem II oxygen-evolving protein (A) and GADPH (B) was determined. Compared with the abundance of 2-week-old seedling sample, increased proteins after the time interval are shown.

study, photosystem II oxygen-evolving protein and GAPDH, are strongly associated with leaf sheath elongation. This result means that photosynthesis and glycolysis are, at least indirectly, GA-responsive. Comparison of Two Proteomics Techniques for Identifying Proteins Regulated by Gibberellin in Rice. By 2D-LC and by 2D-DIGE 1248 protein fractions and 1500 proteins were resolved, respectively. Out of these proteins, 2D-LC identified 9 proteins that were up-regulated and 9 proteins that were down-regulated by GA3 treatment, while 2D-DIGE identified 4 up-regulated and 4 down-regulated proteins. In our previous studies, 32 proteins24 and 8 proteins20 were found to be regulated by GA in leaf sheath elongation. Shen et al. 24 studied leaf sheath segments treated with GA3 in a Petri dish. Tanaka et al.20 used leaf sheath from intact rice seedlings treated with GA3. Although 32 proteins were detected using leaf sheath segments, they were mainly stress-related proteins. In the case of the intact treatment,20 only 5 of the proteins were within the normal range of IEF; the other 3 migrated to the basic side

The 2D-LC technique has been developed to improve quantitative comparisons of protein mixtures in the absence of 2D-PAGE and is the preferred method for detecting low molecular weight proteins. However, poor reproducibility and the large number of replicates required to establish statistical significance are problems that still must be resolved. On the other hand, the 2D-DIGE technique can make exact quantitative comparisons and is very sensitive. Therefore, to study the mechanism of GA action, both 2D-LC and 2D-DIGE methods, along with other proteomic methodology, are important. Such studies will provide us with increasing knowledge about the regulation of agronomically important traits and accelerate the breeding of crops with high productivity, good quality, and broad stress resistance. Abbreviations: 2D, two-dimensional; PAGE, polyacrylamide gel electrophoresis; LC, liquid chromatography; 2D-DIGE, fluorescence two-dimensional difference gel electrophoresis; MS, mass spectrometry; IEF, isoelectric focusing; PVDF, poly(vinylidene difluoride); GA, gibberellin.

Acknowledgment. The authors are grateful to Dr. S. Shen for his technical help. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences and by a grant from a MAFF Rice Genome Project (Japan). References (1) Pandy, A.; Mann, M. Nature 2000, 405, 837-846. (2) Rossignol, M. Curr. Opin. Biotechnol. 2001, 12, 131-134.

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