Proteomic Analysis of Rice Leaf Sheath during Drought Stress Ghulam Muhammad Ali and Setsuko Komatsu* National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Received August 31, 2005
Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice. To investigate the initial response of rice to drought stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to drought conditions from 2 to 6 days, and proteins were extracted from leaf sheaths, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 2 to 6 days, 10 proteins increased in abundance and the level of 2 proteins decreased. The functional categories of these proteins were identified as defense, energy, metabolism, cell structure, and signal transduction. In addition to drought stress, accumulations of protein were analyzed under several different stress conditions. The levels of an actin depolymerizing factor, a light harvesting complex chain II, a superoxidase dismutase and a salt-induced protein were changed by drought and osmotic stresses, but not cold or salt stresses, or abscisic acid treatment. The effect of drought stress on protein in the leaf sheaths of drought-tolerant rice cultivar was also analyzed. The light harvesting complex chain II and the actin depolymerizing factor were present at high levels in a drought-tolerant rice cultivar before stress application. With drought stress, actin depolymerizing factor was expressed in leaf blades, leaf sheaths, and roots. These results suggest that actin depolymerizing factor is one of the target proteins induced by drought stress. Keywords: actin depolymerizing factor • drought • leaf sheath • rice • proteomics
Introduction Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice.1 Drought tolerance is required when plants experience prolonged soil water deficit. Tolerant plants either maintain the water content of tissues, survive a reduction in tissue water content, or recover more completely after rewatering.2 Stomatal closure and osmotic adjustment3,4 are among the plant responses that limit water loss. When plants are subjected to drought stress, rapid stomatal closure, triggered by abscisic acid (ABA), is induced to decrease water loss from the leaves and decreases the availability of CO2 to the photosynthetic apparatus.5 These conditions lead to an overall reduction of the photosynthetic electron transport chain6 that induces oxidative stress.7 In addition, the glycolate oxidase pathway that produces H2O2 is activated during drought stress.8 The effect of drought stress on plants in terms of physiological changes has been well investigated. The responses include stomatal closure, reduced photosynthetic activity and droughtinduced oxidative stress.9,10 Ultrastructural changes in cells and organelles in rice as a result of drought have also been noted.11,12 In wheat leaves, sucrose, proline and quaternary ammonium derivatives are the principal osmotic components.13 * To whom correspondence should be addressed. Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. Tel: 81-29-838-7446. Fax: 81-29-838-7408. E-mail:
[email protected].
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Published on Web 01/04/2006
Chaperone synthesis and oxidative stress tolerance allow plants to survive water deficit.14 Re-watering following drought stress results in leaves that attain a smaller size at maturity and leads to a complete alteration in growth dynamics, carbohydrate and amino acid content, and changes in sink-source related enzymes that helps plants to resume growth;15 however, only a few studies have been undertaken to describe global changes in drought-induced genes16 or proteins17-19 on a genomic scale. On the other hand, rice (Oryza sativa L.) is one of the most important crops in the world. It is the main staple food of more than half of the world’s population.20 Since rice has a genome significantly smaller than those of other cereals, it is an ideal model plant for genetic and molecular studies.21 The draft sequences of rice genomes have been reported for Oryza sativa L. ssp. indica22 and Oryza sativa L. ssp. japonica.23 Furthermore, the complete map-based genome sequence of chromosome 1 (24) and chromosome 4 (25) for Oryza sativa L. cv. Nipponbare have been reported. The challenge ahead for the plant research community is to identify the functions, modifications, and regulatory pathways of proteins encoded by genes. The understanding of the biological function of the novel genes is a more difficult proposition than obtaining just the sequences. This challenge is because the amount of information on amino acid sequences of known proteins in the database does not match the wealth of information on nucleotide sequences being generated through genome projects.26 The analysis of proteins using high-resolution two-dimensional polyacrylamide gel 10.1021/pr050291g CCC: $33.50
2006 American Chemical Society
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Rice Leaf Sheath during Drought Stress
electrophoresis (2D-PAGE) is the most direct approach for defining gene function. Here, we focus on proteomics as a tool for the comprehensive analysis of plant response to drought stress. In the rice leaf blade, four novel drought-responsive mechanisms were revealed using proteome analysis; up-regulation of an S-like RNase homologue, an actin depolymerizing factor and RuBisCO activase, and down-regulation of an isoflavone reductase-like protein.17 The leaf sheath is the main part of the plant that plays a major role in transporting all essential elements with water from the roots to aerial portions of the plant. The leaf sheath supports and stiffens the plant against environmental conditions. Most of the previous studies were carried out using leaf blades to investigate the different stress response on rice.27 In this study, proteome analysis was carried out to investigate the effect of drought on rice leaf sheaths.
Experimental Procedures Plant Materials and Treatment. Rice (Oryza sativa L. cvs. Nipponbare and Zhonghua 8) seedlings were grown in growth chamber. Same amount of soil was added in each pot for all treatments. Two-week-old seedlings were transferred in incubator with holding water to initiate drought conditions, pots were weighed before starting drought condition and at the time of sampling to record water loss each treatment. Protein Extraction. A portion (300 mg) of the excised leaf sheath was homogenized with 1 mL of phosphate buffer28 using a glass mortar and pestle on ice. The homogenate was centrifuged at 15 000 × g at 4 °C for 10 min, and 50% trichloroacetic acid was added to the supernatant to final concentration of 10%. The solution was kept for 30 min on ice and centrifuged at 15 000 × g at 4 °C for 10 min. The resultant precipitate was washed with 100 µL iced ethanol, suspended in 100 µL lysis buffer containing 8 M urea, 2% Nonidet P-40, 2% Ampholine (pH 3.5-10.0; Amersham Biosciences, Piscataway, NJ), 5% 2-mercaptoethanol and 5% poly(vinylpyrrolidone)-40, and sonicated for 2 min twice. The solution was centrifuged at 15 000 × g for 5 min, and 90 µL of the supernatant was subjected to 2D-PAGE. Two-Dimensional Polyacrylamide Gel Electrophoresis. Prepared protein samples (300 µg) were separated in the first dimension by isoelectric focusing (IEF) tube gel and in the second dimension by SDS-PAGE. IEF tube gel solution consisted of 8 M urea, 3.5% polyacrylamide, 2% NP-40, 2% Ampholines (pH 3.5-10.0 and pH 5.0-8.0), ammonium persulfate, and TEMED.29 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 gel. The gels were stained with Coomassie brilliant blue (CBB), and the image analysis was performed. Images of 2D-PAGE were synthesized and the positions of individual proteins on the gels were evaluated automatically using ImageMaster 2D Elite software (Amersham Biosciences). The isoelectric point (pI) and relative molecular mass (Mr) of each protein were determined using 2D-PAGE Markers (Bio-Rad, Hercules, CA). Image Acquisition and Data Analysis. The CBB stained 2-DE gels were scanned in scanner. These images were analyzed with Image Master 2D-Elite software. Image analysis includes the following procedures, which were spot detection, spot measurement, background subtraction and spot matching. Prior to performing spot matching between gel images, one gel image was selected as reference gel. After automatic matching, the
unmatched spots of the member gels were added to the reference gel. The amount of a protein spot was expressed as the volume of that spot which was defined as 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. Cleveland Peptide Mapping. Following separation by 2DPAGE, gel pieces containing protein spots were removed and the protein was electroeluted from the gel pieces using an electrophoretic concentrator (ISCO, Lincoln, CA) at 2 W constant powers for 2 h. After electroelution, the protein solution was dialyzed against deionized water for 2 days and lyophilized. The protein was dissolved in 20 µL of SDS sample buffer (pH 6.8) and applied to a sample well in an SDS-PAGE gel and the sample solution was overlaid with 20 µL of 1/2 SDS sample buffer containing 50 ng/µL Staphylococcus aureus V8 protease (Pierce, Rockford, IL). Electrophoresis was performed until the sample and protease were stacked in the stacking gel, interrupted for 30 min to digest the protein, and then continued.30 N-Terminal and Internal Amino Acid Sequencing. Following separation by 2D-PAGE or by Cleveland method, the proteins were electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane (Pall, Port Washington, NY) using a semidry transfer blotter (Nippon Eido, Tokyo, Japan), and detected by CBB staining. The stained protein spots were excised from the PVDF membrane and applied to a gas-phase protein sequencer Procise cLC (Applied Biosystems, Foster City, CA). The amino acid sequences obtained were compared with those of known proteins in the Swiss-Prot, PIR, Genpept and PDB databases with Web-accessible search program FastA.
Results and Discussion Protein Expression in Rice Leaf Sheaths under Drought Conditions. Chloroplasts are present in both the bundle sheath and mesophyll cells of rice plants. The bundle sheath of C3 plants is considered to have an adaptive role to arid conditions.31 Therefore, it has been proposed that the chloroplasts in bundle sheath cells are more tolerant to drought than the chloroplasts in mesophyll cells. To assess the initial drought response of rice leaf sheaths, changes in protein accumulation were examined after increasingly longer drought periods. Twoweek-old rice seedlings were kept in soil with a water content of about 50-60% for the control plant. In stress treatments, the minimum water content value was 10% at 2, 3, 4, 5, and 6 days. Proteins were extracted from leaf sheaths, separated by 2D-PAGE and stained by CBB (Figure 1). Digital image analysis of the CBB stained gel identified 698 proteins in rice leaf sheaths. Thirty-five proteins responded at 6 days after drought stress by up- or down-regulation. From this population of 35 proteins, a subset of 12 proteins clearly responded to drought as early as 2-5 days after stress application. The levels of 10 proteins were elevated and 2 proteins declined in abundance as a result of drought stress (Figure 1). The 12 proteins that responded to drought by up- or down-regulation were selected for amino acid sequencing. Analysis of Drought Responsive Proteins. To analyze the drought responsive proteins, two-week-old seedlings were exposed to drought conditions for 2 days. Proteins whose levels were altered by drought stress were excised, purified from 2DPAGE gels, and subjected to Edman sequencing to determine Journal of Proteome Research • Vol. 5, No. 2, 2006 397
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Figure 1. Protein responses in rice leaf sheath exposed to different days of drought stress. Rice two-week-old seedlings were treated with drought for 0, 2, 3, 4, 5, and 6 days. Proteins were extracted from leaf sheath, separated by 2D-PAGE and stained by CBB. Following scanning, the gel patterns were analyzed using 2D-Elite software. Black circles are up-regulated proteins and white circles are downregulated proteins. The experiment was repeated five times with similar results.
the amino acid sequences (Table 1). Twelve proteins responding to drought by up- (spots 1, 2, 3, 4, 5, 6, 8, 9, 10, and 11) or down- (spots 7 and 12) regulation were selected for amino acid sequencing. The sequenced proteins were identified as a chloroplast ATPase, serine hydroxymethyltrasferase I, RuBisCO small subunit, a superoxide dismutase (SOD), a salt induced protein (SALT), RuBisCO large subunit, photosystem II oxygen evolving complex protein, oxygen evolving enhancer protein 2, and proteins deduced from DNA sequences of Oryza sativa chromosomes: actin depolymerizing factor, light harvesting complex chain II, phosphoglucomutase cytoplasmic 2, and 2-Cys peroxiredoxin BAS 1. The functional categories of the 12 proteins were identified as defense, energy, metabolism, cell structure, signal transduction and functional unknown protein.32 Down-Regulated Proteins by Drought Stress. In this study, RuBisCO small and large subunits were reduced in leaf sheaths by drought stress (Table 1). Yamane et al.12 investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. The RuBisCO content in bundle sheath chloroplasts was reduced more dramatically than in mesophyll chloroplasts by drought stress. This report and our result suggest that chloroplasts in bundle sheaths are sensitive to 398
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drought stress, and the RuBisCO content is reduced due to chloroplast stress. Up-Regulated Proteins by Drought Stress. In this study, SALT, SOD, light harvesting complex chain II, photosystem II oxygen evolving complex protein, oxygen evolving enhancer protein 2, chloroplast ATPase, serine hydroxymethyltrasferase I, 2-Cys peroxiredoxin, actin depolymerizing factor, and phosphoglucomutase cytoplasmic 2 were enhanced by drought stress (Table 1). SALT is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice roots in response to NaCl stress.33 Also, SALT expression was up-regulated following treatment with a fungal elicitor, jasmonic acid and ABA. SALT protein was localized to xylem parenchyma cells in the vascular bundles of the major and minor leaf veins.33 Our result suggests that SALT is induced not only by salt stress, but also drought stress. Rebeille et al.34 reported that glycine decarboxylase, serine hydroxymethyltransferase, and tetrahydrofolate polyglutamates form a complex in pea leaf mitochondria. The binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. Once bound to the proteins, tetrahydrofolate, a
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Rice Leaf Sheath during Drought Stress Table 1. Identification of Drought Responsive Proteins in Leaf Sheatha,b,c spot no.
Mr
pI
v1
71
5.3
N-VLFSVTKKAT
v2 v3 v4 v5
40 52 27 56
5.6 5.1 5.1 6.7
v6
27
4.7
N-EGVPPXLTFD N-MRTNPTTSRP N-AYGEAANVFG N-blocked I-AFRLDPAKWG N-AGGVDDAPLV
V7 v8
17 23
6.4 4.7
v9
23
5.4
v 10 v 11 V 12
21 17 50
5.4 4.7 6.0
homologous protein (homology %)
sequences
N-XQVWPIEGIK N-blocked I-LTASLPADG N-AEEEAAAPPP N-ATKKAVAVLK N-TLVKIGPWGG N-blocked I-MTLGFVDLLR
accession no.
ratio
Oryza sativa chromosome (Phosphoglucomutase cytoplasmic 2) (100) Photosystem II oxygen evolving complex protein (80) Chloroplast ATPase (100) Oxygen evolving enhancer protein 2 (100) Serine hydroxylmethyltransferase I (100)
AC082645 (AK099746) P14226 X3396 P81668 AF439728
+1.5
Oryza sativa chromosome (2-Cys peroxiredoxin BAS1) (100) RuBisCO small chain (100) Oryza sativa chromosome (Purative actin depolymerizing factor) (100) Oryza sativa chromosome (Light harvesting complex chain II, fragment) (100) Superoxide dismutase (100) Salt induced protein (100) RuBisCO large chain (100)
AP004753 (AK104703) A05005 AC104433 (XP470137.1) AP005200 (PT0037) S29146 AF001395 P25413
+2.3
+1.5 +2.1 +2.0 +1.8
-1.5 +2.3 +1.9 +2.0 +2.9 -1.6
a N: N-terminal amino acid sequence and I: Internal amino acid sequence. b v: Up-regulated proteins and V: Down-regulated proteins. c Ratio: Amount of target protein in control versus samples treated by drought for 2 days.
very O2-sensitive molecule, was protected from oxidative degradation.34 This report and our results suggest that serine hydroxymethyltransferase is induced for protection from oxidative degradation by drought stress. Actin-binding proteins are classified into several functional categories, including profilins, formins, actin depolymerizing factors/cofilins, and cyclase-associated proteins.35 Actin depolymerizing factors are known to be induced by salt, drought and cold stresses in cereal plants,17,36 suggesting that actin depolymerizing factor might be required for osmo-regulation under osmotic stress. Indeed, osmotic stress regulation of actin organization correlated well with K+ channel activity in guard cells.37 This result indicates that osmotic stress caused by drought may subsequently induce actin depolymerizing factors in rice seedlings.38 In the absence of sufficient CO2 as the ultimate electron acceptor, electrons flow from the photosynthetic membrane to oxygen molecules via the Mehler reaction and create superoxide ions.39 These ions are converted into less toxic hydrogen peroxide molecules by the various SODs that are distinguished by subcellular location (chloroplast, cytosol, mitochondrion, and peroxisome) and by the metal at the active site (Cu-Zn, Mn, and Fe). Kaminaka et al.40 and Salekdah et al.17 reported that SOD genes and SOD proteins were upregulated in rice by drought stress and ABA. The hydroxyl radical is generated from H2O2 by the Fe (II)-catalyzed Fenton reaction and has been implicated in the nonenzymic fragmentation of the RuBisCO large subunit in wheat chloroplasts.41 Our results show also that SOD was up-regulated and RuBisCO was down-regulated in rice as a result of drought stress. Drought could involve CO2-sensitive modification of photosynthetic metabolism depending on plant growth conditions and possibly also on plant species.42 On the other hand, engineering the feedback de-excitation capacity by the 22-kDa photosystem II subunit, a member of the light harvesting complex protein super family, by overexpression could potentially yield crop plants that are more resistant to environmental stress.43 Feedback de-excitation might be regulated to prevent inhibition of photosynthesis, and in our experiment the level of light harvesting complex chain II increased with drought treatment.
Huo et al.44 reported that using a salt-tolerant mutant of wheat, 5 candidate proteins were identified in the chloroplast as being responsible for salt tolerance. The proteins are a H+transporting two-sector ATPase, a glutamine synthase 2 precursor, an oxygen evolving protein of photosystem II and RuBisCO small subunit. These proteins are likely to play a crucial role in maintaining the function of the chloroplast and the whole cells when the plant was under stress. A 2-Cys peroxiredoxin, which is a nuclear-encoded chloroplast protein that decreases oxidative damage to chloroplast proteins,45 was up-regulated by drought in rice seedling. Haiheidari et al.19 was also reported that 2-Cys peroxiredoxin was up-regulated in sugar beet leaves under drought stress. The chloroplast is particularly prone to oxidative damage by photosynthetic oxygen production and activation. The chloroplastic ascorbate peroxidase-dependent water/water cycle is highly sensitive to inactivation by ROS and is often insufficient to protect the photosynthetic apparatus from photoinhibition during severe drought stress.46 Therefore, the 2-Cys peroxiredoxin-dependent water/water cycle can be an important alternative metabolic pathway to detoxify H2O2 under normal conditions as well as under drought-induced oxidative stress. Phosphogucomutase catalyzes the interconversion of glucose1- and glucose-6-phosphate in the synthesis and consumption of sucrose. Manjunath et al.47 reported that phosphogucomutase is a stable protein and that existing levels are sufficient to maintain the flux of glucose-1-phosphate into glycolysis under O2 deprivation. In yeast, phosphoglucomutase cytoplasmic 2 was induced by heat-shock and salt stresses.48 In this study, phosphogucomutase cytoplasmic 2 was also up-regulated by drought in rice seedling. Protein Expression under Different Stresses. The effect of different stress conditions on protein accumulation in rice leaf sheaths was determined. Rice seedlings were treated with drought by withholding water, 300 mM mannitol as an osmotic stress, 150 mM NaCl as a salt stress, cold at 5 °C, and 50 µM ABA for 2 days. Proteins were extracted from leaf sheaths, separated by 2D-PAGE and stained by CBB (Figure 2). After digital image analysis, 12 proteins showed reproducible changes in accumulation as a result of drought stress: 7 proteins were changed by salt stress, 8 proteins by osmotic stress, 6 proteins Journal of Proteome Research • Vol. 5, No. 2, 2006 399
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Figure 2. Changes in protein abundance to different stresses. Two-week-old seedlings were treated with drought by with holding water, 300 mM mannitol as osmotic stress, 150 mM NaCl as salt stress, cold at 5 °C, and 50 µM ABA for 2 days. Proteins were extracted from leaf sheath, separated by 2D-PAGE and stained by CBB. Solid line circles are changed proteins by drought and osmotic stresses. Dot line circles show that proteins were not changed by salt, cold and ABA stresses. Spots numbers indicate the same proteins in Figure 2
by cold stress and 8 proteins by ABA treatment. Out of them, actin depolymerizing factor (spot 8), light harvesting complex chain II (spot 9), SOD (spot 10) and SALT (spot 11) were induced by drought and osmotic stresses, but did not change with salt stress, or cold stress and/or ABA treatment. Actin depolymerizing factor (Salekdeh et al., 2002), light harvesting complex chain II42, SOD,17 and SALT33 have also been reported to be induced by environmental stresses; however, in this study these proteins did not change because only bundle sheaths were used. Drought Effects on Proteins in Leaf Sheaths of Drought Tolerant Varieties. A comparative study on morphology and the proteome was carried out between Nipponbare and the drought-tolerant cultivar, Zhonghua 8. Water was withheld for 2 days from two-week-old seedlings of Nipponbare and Zhonghua 8 followed by normal watering for 4 days. Under drought stress, the growth of both cultivars was repressed (Figure 3, upper and left). Shoot elongation was obviously inhibited, and the leaf blade gradually withered from the top to the bottom. The damage to rice seedlings of Nipponbare was more severe than Zhonghua 8; however, after rewatering, Zhonghua 8 400
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suffered less from drought stress than Nipponbare (Figure 3, upper and light). Proteins from the leaf sheaths were extracted, separated by 2D-PAGE and stained by CBB. From these two cultivars, the effect of stress on protein profiles in the leaf sheath of drought-tolerant rice was investigated. Protein patterns from Nipponbare and Zhanghua 8 were similar (Figure 3, lower). Actin depolymerizing factor (spot number 8), photosystem II oxygen evolving complex protein (spot number 2), oxygen evolving enhancer protein 2 (spot number 4) and light harvesting complex chain II (spot number 9) were present in high levels in the drought-tolerant rice cultivar, Zhanghua 8 (Figure 3, lower), suggesting that these proteins help to confer resistance to environmental stresses. Indeed, Li et al.42 have reported that plants overexpressing the light harvesting complex chain II were more resistant to environmental stress. Tissue-Specific Expression of Drought Responsive Proteins. The profiles of drought-responsive proteins found in rice leaf sheaths were compared with other parts of the rice plant. Twoweek-old seedlings were treated without or with drought for 2 days. The proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2D-PAGE and stained by CBB (Figure
Rice Leaf Sheath during Drought Stress
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Figure 3. Comparative response of rice cultivars to drought stress. Two-week-old seedlings from Nipponbare and Zhonghua 8 were treated with drought with holding water for 2days (upper and left), and then were rewatered for 4 days (upper and right). Proteins were extracted from leaf sheath of seedlings without or with drought treatment for 2 days, separated by 2D-PAGE and stained by CBB. Circles are changed proteins by stress. Spots number indicates the same proteins in Figure 2.
4). To determine the relative Mr and pI of each protein, 2DPAGE markers were included. RuBisCO small subunit (spot 7), RuBisCO large subunit (spot 12), an actin depolymerizing factor (spot 8) and SALT (spot 11) were detected in leaf blades, but they did not change as a result of drought stress as they did in leaf sheaths. On the other hand, actin depolymerizing factor (spot 1) was up-regulated by drought stress in both leaf blades and leaf sheaths. In roots, a functionally unknown protein (spot 1) and an actin depolymerizing factor (spot 8) increased with drought treatment in a manner similar to the response by leaf sheaths. Actin depolymerizing factor was detected in all organs of rice plants and increased with drought and osmotic stresses,
suggesting that this protein might be required for osmoregulation under osmotic stress. Indeed, osmotic stress regulation of actin organization correlates well with K+ channel activity in guard cells.37 Depolymerization of actin filaments by osmotic stress potentiates the inward K+ current in guard cells.37 Therefore, actin filaments may serve as an osmosenser and target inward K+ channels in guard cells for regulation. Yan et al.49 reported that an actin-binding protein that is classified as an actin depolymerizing factor increased rice root with salt stress. In our study, the actin depolymerizing factor did not increase with salt stress, suggesting that only certain actin binding proteins may be specifically induced by drought or osmotic stress. These results suggest that actin depolymerizing Journal of Proteome Research • Vol. 5, No. 2, 2006 401
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Figure 4. Protein changes showing tissue specific expression under drought stress. Two-week-old seedlings were treated without or with drought for 2 days. The proteins were extracted from leaf blade, leaf sheath and root, separated by 2D-PAGE and stained by CBB. Circles are changed proteins by stress. Spots numbers indicate to same proteins in Figure 2. The pI and Mr of each protein was determined using 2D-PAGE markers (Bio-Rad)
factor, detected in this study, is one of the target proteins induced by drought stress.
Concluding Remarks This study gives new insights into the drought stress response in rice and demonstrates the power of the proteomic approach in plant biology studies. This study also indicates that only certain actin-binding proteins may be specifically induced by drought or osmotic stress. These results suggest that actin depolymerizing factor, detected in this study, is one of the target proteins induced by drought stress. There are several immediate extensions of our work that will increase our understanding of drought responsiveness in rice and may lead to applications in breeding for enhanced drought tolerance. Abbreviations. 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; PVDF, poly(vinylidene difluoride); CBB, Coomassie brilliant blue; ABA, abscisic acid; SOD, superoxidase dismutase; SALT, salt induced protein.
Acknowledgment. We thank Dr. S. Shen for providing Zhonghua 8. We thank the JIRCAS for the scholarship grant awarded to Dr. G. Ali. 402
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