JXB Advance Access published online on January 8, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl226
Integrated Approaches to Sustain and Improve Plant Production under Drought Stress Special Issue |
Proteome response of Elymus elongatum to severe water stress and recovery
Agricultural Biotechnology Research Institute of Iran (ABRII), PO Box 31535-1897, Karaj, Iran
To whom correspondence should be addressed. E-mail: h_salekdeh{at}abrii.ac.ir or hsalekdeh{at}yahoo.com
Received 29 December 2005; Accepted 9 October 2006
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Abstract |
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Tall wheatgrass (Elymus elongatum Host) is a drought-tolerant, cool-season forage grass native to Iran. A proteomic approach has been applied to identify mechanisms of drought responsiveness and tolerance in plants undergoing vegetative stage drought stress and then recovery after rewatering. Uniformed clones were reproduced from a parent plant collected from Brojen (central region of Iran). Clones were grown in pots and drought was initiated by withholding water for 16 d. The leaf samples were taken in triplicate from both stressed/rewatered plants and continuously watered controls at five times: (i) 75% FC, (ii) 50% FC, (iii) 25% FC, (iv) 3 d after rewatering, and (v) 14 d after rewatering. Changes in the proteome pattern of shoots were studied using two-dimensional gel electrophoresis. Following the 16 d water stress, both shoot dry weight and leaf width decreased up to 67% compared with the well-watered plants, whereas proline content increased up to 20-fold. Leaf relative water contents (RWC) also declined from 85% to 24%. Out of about 600 protein spots detected on any given two-dimensional gel, 58 protein spots were reproducibly and significantly changed during drought stress and recovery. Only one protein (abscisic acid- and stress-inducible protein) showed significant changes in expression and position in response to severe drought. The fifty-eight responsive proteins were categorized in six clusters including two groups of proteins specifically up- and down-regulated in response to severe drought stress. Eighteen proteins belonging to these two groups were analysed by liquid chromatography tandem mass spectrometry leading to the identification of 11 of them, including the oxygen-evolving enhancer protein 2, abscisic acid- and stress-inducible protein, several oxidative stress tolerance enzymes, two small heat shock proteins, and Rubisco breakdown. The results suggest that E. elongatum may tolerate severe drought stress by accumulating proline and several proteins related to drought-stress tolerance. Recovery after rewatering might be another mechanism by which plants tolerate erratic rainfall in semi-arid regions.
Key words: Abscisic acid- and stress-inducible protein (ASR), antioxidant, drought, Elymus elongatum, heat shock proteins, oxygen evolving enhancer protein 2 (OEE2), proteomics, recovery, tall wheatgrass, two-dimensional gel electrophoresis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Drought is one of the major limiting factors of plant production worldwide. Plant adaptation to drought is the result of many different physiological and molecular mechanisms. Several studies have shown that physiological adaptations to water stress were associated with drought-induced proteins (Bray, 1997). Proteomics has proved to be a powerful tool for the identification of proteins and mechanisms involved in drought response and tolerance (Riccardi et al., 1998; Kawasaki et al., 2000; Salekdeh et al., 2002a, b; Hajheidari et al., 2005). Most of these studies were performed on plants under mild water stress. Riccardi et al. (1998) studied the effect of progressive water stress on the leaves of maize. The water potential of stressed leaves was generally between –0.8 MPa and –1.0 MPa in the growth cabinet compared with –1.5 MPa in the greenhouse. Out of 78 responsive proteins, 16 were identified, including proteins involved in the water-stress response, the basic metabolic pathway, and lignification. Kawasaki et al. (2000) examined the responses of leaf proteins of wild watermelon to 3 d of drought stress in small pots. The leaf water potential was maintained at –1.1 MPa compared with –0.7 MPa in control plants and –2 MPa in stressed leaves of drought-sensitive domesticated watermelon. They identified seven newly induced proteins including citrulline, a scavenger of hydroxyl radicals. Salekdeh et al. (2002b) analysed the proteome responses of rice leaves to mild drought stress. Three-week-old plants of rice developed gradual water stress over 23 d, during which period the midday leaf water potential declined to –2.4 MPa, compared with –1.0 MPa in well-watered controls. Studies on mild drought stress in rice leaves showed that drought-induced changes in about 42 proteins were reversed completely within 10 d of rewatering. Mass spectrometry helped to identify 23 of these proteins including proteins involved in basic metabolic pathways such as ATP production, photosynthesis, protein synthesis, oxidative stress tolerance, and cytoskeleton reorganization.
An integrated physiological and proteomic approach has been used here to study the response of tall wheatgrass (Elymus elongatum Host) to severe drought stress and recovery after rewatering. Tall wheatgrass is a cool-season forage grass of the rangelands in Iran that is located on arid and semi-arid regions (FAO, 1997) characterized by low and unpredictable rainfall. Drought tolerance in this species has been reported (Garcia et al., 2002), but their physiological and, in particular, molecular bases have not been well known. Molecular analyses of wild species such as drought-tolerant grasses would provide a better insight into genes and mechanisms by which plants may adapt to prolonged drought.
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Materials and methods |
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Plant growth and treatments
The seeds of tall wheatgrass (Elymus elongatum Host) were collected from the city of Brojen, Markazi province, in Iran and were then sown in the field at the Agricultural Biotechnology Research Institute of Iran in Karaj under normal conditions. The parental clone (genetically homogeneous) of tall wheatgrass was collected at tillering stage from single 2-year-old plants from the field plots and propagated in pots. The clones with three tillers were grown in pots (40 cm deep, 20 cm diameter) filled with a mixture sandy clay loam soil (48.3% sand, 27.2% clay, and 24.5% silt) in a greenhouse for 80 d before beginning the stress treatment. The soil had 19.2 g water per 100 g dry soil at field capacity (FC). The greenhouse environmental conditions were 28/18 °C (night/day), relative humidity of 50–60%, 14 h photoperiod, and a photosynthetically active radiation of 900 µmol m–2 s–1. Drought was initiated by withholding water and drought-stressed pots were rewatered when the soil moisture reached 25% FC. Soil moisture content was monitored to calculate water relationships by weighing the pots at 09.00 h daily. The well-watered treatment was maintained near FC by adding the amount of water lost by evapotranspiration every day. The evapotranspiration from the pot surface was calculated in well-watered and droughted pots. Three pots without plants were used as blanks to measure evaporation and were treated as droughted pots. The leaf samples were collected from well-watered and stressed/rewatered plants at five times: (i) 75% FC (4 d after stress, DAS), (ii) 50% FC (7 DAS), (iii) 25% FC (16 DAS), (iv) 3 d after rewatering (Re 3), and (v) 14 d after rewatering (Re 14) in triplicate. Several physiological traits were measured including leaf relative water content (RWC), shoot length, leaf width (rolling), shoot fresh and dry weights. RWC and proline content were measured as described by Barrs and Weatherley (1962) and Bates et al. (1973), respectively. Shoot dry weight was measured by harvesting the above-ground biomass during the experiment. After harvesting, fresh weight was obtained immediately and then dry weight was measured by drying in an oven at 80 °C for 48 h.
Protein extraction and electrophoresis
Total soluble proteins were extracted from leaves of three independent replications from each treatment according to the methods of Damerval et al. (1986) with some modifications (Salekdeh et al., 2002b). The samples were ground in liquid nitrogen and suspended in 10% (w/v) trichloroacetic acid in acetone with 0.07% (w/v) dithiothreitol (DTT) at –20 °C for 1 h, followed by centrifugation for 15 min at 35 000 g. The pellets were washed with ice-cold acetone containing 0.07% DTT, incubated at –20 °C for 1 h and centrifuged again at 4 °C. This step was repeated three times and then the pellets were lyophilized. The sample powder was then solubilized in lysis buffer [9.5 M urea, 2% (w/v) CHAPS, 0.8% (w/v) Pharmalyte pH 3–10, 1% (w/v) DTT] and the protein concentration was determined by the Bradford assay (Bio-Rad) with BSA as the standard. Isoelectric focusing (IEF) of approximately 120 µg (for preparative gels 1–1.5 mg) total protein was carried out on an immobilized pH gradient 24 cm pH 4–7L strips on Pharmacia Multiphor II (Amersham Pharmacia Biotech). The running condition was as follows: 500 V for 1 h, followed by 1000 V for 1 h, and finally 3500 V for 16 h. The focused strips were equilibrated twice for 15 min in 10 ml equilibration solution. The first equilibration was performed in a solution containing 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT, and 50 mM TRIS–HCl buffer, pH 8.8. The second equilibration was performed in a solution modified by the replacement of DTT by 2.5% (w/v) iodoacetamide. Separation in the second dimension was performed by SDS–PAGE in a vertical slab of acrylamide (12% total monomer, with 2.6% crosslinker) using a Dodeca Cell (Bio-Rad). The analytical 2D gels were stained with silver nitrate as described by Blum et al. (1987) with some modifications.
Staining and image analysis
The silver-stained gels were scanned at a resolution of 600 dots per inch on a GS-800 densitometer (Bio-Rad). The scanned gels were saved as TIF images for subsequent analysis. Spot quantitation was carried out using the Melanie-3 software (GeneBio, Geneva, Switzerland). The parameters for protein spot detection are as follows: number of smoothes, 2; Laplacian threshold, 3; partial threshold, 3; saturation, 90; peakness increase, 100; minimum perimeter, 35. After image treatment, spot detection, protein quantification, and spot pairing were carried out based on Melanie-3 default settings. Then, spot pairs were investigated visually and the scatter plots between gels of each data point were displayed to estimate gel similarity and experimental errors. The molecular masses of proteins on gels were determined by co-electrophoresis of standard protein markers (Amersham Pharmacia Biotech) and pI of the proteins were determined by migration of the protein spots on 18 cm IPG (pH 4–7 linear) strips.
Statistical analyses and protein classification
The pots were arranged as a completely randomized design with three replicates. Student's t test (paired) was used to compare the per cent volume of spots for determining the significance of differences means treatments for each sampling versus the control. The induction factor (IF) was also calculated by dividing the per cent volume of spots in stressed/rewatered gels to the per cent volume of corresponding spots in well-watered samples. Statistical analysis was performed with the SAS 6.12 software (SAS Institute, 1996). Spots were determined to be significantly up- or down-regulation when P 
0.05. Hierarchical clustering was performed using 2.11 software based on the Pearson correlation coefficient (http://rana.lbl.gov/EisenSoftware.htm) (Eisen et al., 1998). The induction factor (IF) of drought-responsive proteins was applied as input data after preprocessing.
Mass spectrometry analysis
Preparative gels were stained with colloidal Commassie Brilliant Blue G-250 (Neuhoff et al., 1988). Protein spots were manually excised from the gels, digested using trypsin, and analysed using a Micromass QTOF Ultima mass spectrometer (Micromass, Manchester, UK) at the Australian Proteome Analysis Facility (APAF). Protein samples were destained and underwent a 14 h tryptic digest at 37 °C. After digestion the gel pieces were extracted initially with 0.1% aqueous formic acid (50 µl) and then with 50 µl H2O:ACN (1:1 v/v), and 0.1% formic acid for 15 min. The combined extracts were dried and the peptides resuspended in 0.1% aqueous formic acid (20 µl). Digested peptides were separated by nano-LC using an Ultimate HPLC and Famos autosampler system (LC-Packings, Amsterdam, Netherlands). Samples (5 µl) were concentrated and desalted onto a micro C18 pre-column with H2O:ACN (98:2 v/v), and 0.1% formic acid at 20 µl min–1. After a 4-min wash the precolumn was switched into line with the analytical column containing C18 RP silica (PEPMAP, 75 µm x15 cm, LC-Packings). Peptides were eluted from the column using a linear solvent gradient from H2O:ACN (95:5 v/v), and 0.1% formic acid to H2O:ACN (40:60 v/v), and 0.1% formic acid at 200 nl min–1 over a 30 min period. The LC eluent was subject to positive ion nanoflow electrospray analysis on an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City, CA). The QStar was operated in information-dependent acquisition mode (IDA). In IDA mode a TOFMS survey scan was acquired (m/z 350–1700, 0.75 s), with the two largest multiple-charged ions (counts >15) in the survey scan sequentially subjected to MS/MS analysis. MS/MS spectra were accumulated for 5 s (m/z 50–2000). A processing script was used to generate data suitable for submission to the database search program (Mascot, Matrix Science http://www.matrixscience.com/). Where Mascot failed to provide identification, spectra were interpreted both manually and using sequencing algorithms, with the derived amino acids sequences subjected to EMBL MS BLAST (http://dove.embl-heidelberg.de/Blast2/msblast.html) homology-based searching.
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Results |
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Physiological response to drought stress
Clones were grown in pots for 80 d prior to water withdrawal for 16 d. During drought treatment, an average of 188 ml water was added to each well-watered pot. Soil water content decreased from 19.2% at FC to 4.8% at 25% FC (Fig. 1a). The total amount of water loss in well-watered, drought-stressed, and blank pots were 2720, 1204, and 553 ml, respectively. The major water loss in droughted pots was observed during days 4–6 (Fig. 1b). Average daily evapotranspiration rates in plants at FC, 75%, 50%, and 25% FC were 170, 162, 66, and 13 ml, respectively.
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Shoot dry weight, leaf width, and RWC remarkably decreased during stress progression. At 25% FC, shoot dry weight and leaf width decreased up to 67.3% and 78.8%, respectively, compared with control plants. Leaf relative water contents (RWC) declined from 85% in well-watered plants to 24% at 25% FC. Leaf proline concentration was increased slightly from 75% FC to 50% FC and then increased up to 20-fold at 25% FC. At 14 d after rewatering, RWC, leaf width, and proline concentration returned to the well-watered levels at 14 d after rewatering, whereas shoot dry weight did not reach the control level even 14 d after rewatering (Fig. 2).
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Two-dimensional gel electrophoresis analysis
Effects of drought stress and rewatering on leaf protein patterns of tall wheatgrass were studied using 2DE. Melanie-3 analysis of at least three replicated gels revealed that 600 proteins were reproducibly detected in silver-stained gels over a pH range of 4–7 and molecular weight range 14–94 kDa (Fig. 3). Fifty-eight spots showed reproducible and significant changes under drought stress and reverted to well-watered levels after rewatering. Among them, 28 protein spots were up-regulated, 29 protein spots were down-regulated, and only one protein spots showed to be present only under stress conditions (spot 264) (Table 1). Only one protein (abscisic acid- and stress-inducible protein) showed significant changes in expression and position (spots 63, 66, and 67) in response to severe drought. The numbers of up-regulated proteins were increased with stress progression and the highest number of up-regulated protein spots was observed at 25% FC (21 spots), whereas, the numbers of down-regulated proteins increased from 75% FC to 50% FC (from 11 to 23 spots) and then decreased (13 spots). After 14 d of rewatering, the expression level of all responsive proteins returned to the well-watered levels (Fig. 4). Hierarchical clustering was applied to categorize responsive proteins as described by Eisen et al. (1998). Fifty-seven responsive protein spots were categorized in six expression groups (Fig. 5). Protein spot 264 which was expressed only in stress condition was not included in this analysis. The largest group was composed of proteins up-regulated only under drought stress. Eighteen of these proteins were analysed using liquid chromatography tandem mass spectrometry (LC/MS/MS).
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Protein identification
Eighteen drought-responsive proteins analysed by LC/MS/MS led to the identification of 11 proteins (Table 2). Protein spot 279, which showed the highest induction ratio was identified as dehydroascorbate reductase. Protein spot no. 264 was detectable only under stress conditions and was identified as a heat shock protein (Table 2). Other proteins were identified as cytosolic Cu-Zn superoxide dismutase (spot 38), glutathione S-transferase (spot 70), glutathione peroxidase (spot 277), oxygen-evolving enhancer 2 chloroplast precursor (spot 281), Rubisco breakdown (spot 280), and heat shock protein (spot 278), and abscisic acid- and stress-inducible protein (spots 63, 66, and 67). The expression patterns of cytosolic Cu-Zn superoxide dismutase (spot 38), dehydroascorbate reductase (spot 279), and heat shock protein (278) were shown in Fig. 6.
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Discussion |
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Tolerant plants are able to maintain tissue water content, or survive a reduction in tissue water content, or recover more completely after rewatering (Cabuslay et al., 1999). Osmotic adjustment and stomatal closure are among the responses that limit water loss. In the present study, drought stress caused both morphological and physiological changes in plants under stress, resulting in a remarkable reduction in shoot dry weight (67.3%). Ninety per cent of total evapotranspiration in the plants subjected to water withholding had occurred before the soil moisture reached 50% FC, indicating a rapid closure of stomata in response to stress. However, the leaf RWC dropped down to about 24%, showing a failure in maintaining tissue water content.
The level of proline increased up to 20-fold in response to severe drought conditions. The proline accumulation up to 10-fold in velvet grass (Holcus lanatus L.) and 35-fold in Ber (Ziziphus mauritiana Lamk.), a drought-tolerant tree, after exposure to severe water stress have also been reported (Pedrol et al., 2000; Clifford et al., 1998). Proline is considered as a compatible solute (Samaras et al., 1995) and an osmoprotectant (Serrano and Gaxiola, 1994; Okuma et al., 2000). Proline may confer a protective effect by protecting or inducing stress-protective proteins (Khedr et al., 2003). They reported that severe salt-stress caused an inhibition of the antioxidative enzymes catalase and peroxidase in Pancratium maritimum L., but the activity of these enzymes was also maintained significantly higher in the presence of proline. They concluded that proline improves the salt-tolerance of Pancratium maritimum L. by both protecting the protein turnover machinery against stress-damage and up-regulating stress protective proteins. The high level of proline under severe stress in our experiment might improve plant tolerance through the regulation of stress-responsive genes and its osmoprotective roles. Fourteen days after rewatering, all measured traits but shoot dry weight returned to the well-watered level, indicating a partial recovery after rewatering.
Cluster analysis of drought-responsive proteins revealed that a large number of proteins are up-regulated only under severe drought stress (25% FC) (Fig. 5). Mass spectrometry analysis of 18 of these proteins led to the identification of 11 proteins. Of them, seven proteins were identified as chaperone proteins and oxidative defence enzymes which allow plants to survive water deficit (Smirnoff, 1998). As a result of stomatal closure and 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 (Noctor et al., 2002). The enhanced amount of ROS can be viewed as a threat for the cell and as secondary messengers involved in the stress-response signal transduction pathway. Therefore, plant cells require two different mechanisms which will enable the detoxification of excess ROS and fine modulation of ROS for signalling purposes.
SOD acts as a first line of defence converting superoxide to the less toxic hydrogen peroxide molecules (Alscher et al., 2002). The abundance of the cytosolic Cu-Zn SOD of E. elongatum increased up to 2-fold in response to drought. Cytosolic Cu-Zn SOD was shown to be up-regulated in rice (Salekdeh et al., 2002b) and sugar beet (Hajheidari et al., 2005). The up-regulation of this enzyme in plants grown under progressive stress reveals its important role in response to drought.
The detoxification of H2O2 is accomplished with ascorbate peroxidase, glutathione peroxidase, catalase, and 2-Cys peroxiredoxin. Ascorbate peroxidase reduces H2O2 to water, with the concomitant generation of monodehydroascorbate (MDHA). MDHA is a radical with a short lifetime that disproportionates to ascorbate and dehydroascorbate (DHA). DHA is reduced to ascorbate by the action of DHA reductase, using glutathione as the reducing substrate. The most up-regulated protein (4.8-fold) was identified as dehydroascorbate reductase (spot 279) suggesting its strong role in the detoxification of H2O2.
The expression of glutathione peroxidase (spot 277) and glutathione-S-transferases increased up to 2.12- and 1.7-fold, respectively, at 25% FC compared with well-watered plants. The enzymes glutathione-S-transferases have been associated with both normal cellular metabolism as well as in the detoxification of xenobiotics, limiting oxidative damage and other stress responses in plants. The endogenous products of oxidation including membrane lipid peroxides and products of oxidative DNA degradation are highly cytotoxic. The glutathione-S-transferases detoxify such endogenously produced electrophiles by their conjugation with GSH. Both GSTs and glutathione peroxidases can also detoxify such products directly (Marrs, 1996). H2O2 is a key regulatory molecule in the response to stresses (Mittler, 2002), and its ability selectively to induce a subset of defence genes like glutathione-S-transferases and glutathione peroxidases without directly inducing other defence genes. The up-regulation of both glutathione-S-transferases and glutathione peroxidase may represent such co-expression mechanisms. One effect of transpiration is to cool the leaves and once the rate of transpiration decreases, plants suffer from multiconstraints including increasing temperature (Yokota et al., 2002). Two small heat shock proteins (sHSPs) that were induced in E. elongatum shoots after progressive water deficit are described. These proteins have been reported to be involved in protecting macromolecules like enzymes, lipids, nucleic acids, and mRNAs from dehydration (Yamaguchi-Shinozaki et al., 2002). Wehmeyer et al. (1996) suggested that sHSPs are among several factors required for desiccation tolerance. Hajheidari et al. (2005) reported the up-regulation of two sHSP under drought stress in sugar beet grown in the field.
Protein spots 63, 66, and 67 with close pI and MW were up-regulated up to 2.2, 2.4, and 4.2 under sever drought conditions (Fig. 7). These proteins showed sequence homology with abscisic acid- and stress-inducible protein (ASR). The up-regulation of this gene has been also reported in response to drought stress in maize (Riccardi et al., 1998) and salt stress in a salt-tolerant rice genotype (Salekdeh et al., 2002a). The function of the ASR1 proteins is not understood, but they may be involved in the protection of DNA structure during osmotic stress. Iusem et al. (1993) has reported that characterization of cDNA encodes ASR protein whose expression was activated by leaf water deficit and fruit ripening. The results of subcellular fractionation experiments revealed that the protein is located primarily in the nucleus. Silhavy et al. (1995) identified a nuclear-targeting sequence motif in this gene isolated from Solanum chacoense Bitter which is extremely resistant to viruses, insects, and drought. They suggested that this basic protein (pH 7.9) may be involved in the protection of DNA structure during water loss or in gene regulation upon stress by changing DNA topology. The three different forms of ASR may represent different forms of this protein that may differ in a number of phosphoryl groups as explained previously by Salekdeh et al. (2002a). The expression pattern of three proteins at different stages suggests possible changes in both expression and phosphorylation levels in response to severe stress.
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The expression level of OEE2 increased up to 2.6-fold in response to severe drought stress. Murota et al. (1994) reported a role for OEE2 in salt adaptation in photoautotrophically cultured green tobacco cells. Increased expression of OEE2 under salt stress has been also reported in mangrove and rice (Sugihara et al., 2000; Abbasi and Komatsu, 2004). It is known that OEE2 can be easily removed from the PSII complex in the presence of NaCl. The increased expression levels of OEE2 might be needed to repair protein damage caused by dissociation and to keep oxygen evolving.
An increase in abundance of Rubisco breakdown was also observed, which has already been reported in rice (Salekdeh et al., 2002b) and sugar beet (Hajheidari et al., 2005) under drought stress. It is possible that some fragmentation occurs in vitro during protein solubilization. The increased abundance of protein fragments may have resulted from in vitro breakdown of protein generated by ROS or occur in vitro during protein solubilization.
In conclusion, the protein expression pattern of E. elongatum leaves is described and their possible roles in adaptation to drought stress and recovery are discussed. The combination of proteomic and physiological studies provided us with a better understanding of drought-tolerance mechanisms in this plant. Our results suggest that E. elongatum may tolerate severe drought stress by surviving the reduction in tissue water content through the up-regulation of several oxidative stress-tolerance enzymes, small heat shock proteins, OEE2, and ASR. Recovery after rewatering might be another mechanism by which plants tolerate erratic rainfall in semi-arid regions. More work is needed to determine if increased expression provides a growth advantage, or if higher levels are a symptom of greater stress injury.
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Footnotes |
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* These two authors contributed equally in this paper.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abbasi FM and Komatsu S. (2004) A proteomic approach to analyse salt-responsive proteins in rice leaf sheath. Proteomics 4 2072–2081.[CrossRef][ISI][Medline]
Alscher RG, Erturk N, Heath LS. (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany 53 1331–1341.
Barrs HD and Weatherley PE. (1962) A re-examination of the relative turgidity techniques for estimating water deficits in leaves. Australian Journal of Biology Science 15 413–428.
Bates LS, Waldren RP, Teare LD. (1973) Rapid determination of free proline for water-stress studies. Plant and Soil 39 205–207.[CrossRef][ISI]
Blum H, Beier H, Gross HJ. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8 93–99.[CrossRef][ISI]
Bray EA. (1997) Plant responses to water deficit. Trends in Plant Science 2 48–54.
Cabuslay G, Ito O, Alejar A. (1999) Genotypic differences in physiological responses to water deficit in rice. In O'Toole J, Ito O, Hardy B (Eds.). Genetic improvement of rice for water-limited environmentsLos Baños International Rice Research Institute pp. 99–116.
Clifford SC, Arndt S, Corlett JE, Joshi S, Sankhla N, Popp M, Jones HG. (1998) The role of solute accumulation, osmotic adjustment and changes in cell wall elasticity in drought tolerance in Ziziphus mauritiana (Lamk.). Journal of Experimental Botany 49 967–977.
Damerval C, de Vienne D, Zivy M, Thiellement H. (1986) Technical improvement in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling protein. Electrophoresis 7 53–54.
Eisen MB, Spellman PT, Brown PO, Botstein D. (1998) Cluster analysis and display of genome-wide expression patterns. Proceeding of the National Academy of Sciences, USA 95 14863–14868.
FAO (Food and Agriculture Organization). (1997) Statistical database. The state of Iran Agri-Food country profile. Rome. http://www.fao.org/ag/agl/aglw/aquastat/contries/iran.
Garcia MG, Busso CA, Polci P, Garcia Girou NL, Echenique V. (2002) Water relations and leaf growth rate of three Agropyron genotypes under water stress. Biocell 26 309–317.[ISI][Medline]
Hajheidari M, Abdollahian-Noghabi M, Askari H, Hedari M, Sadeghian SY, Ober ES, Hosseini Salekdeh GH. (2005) Proteome analysis of sugar beet leaves under drought stress. Proteomics 5 950–960.[CrossRef][ISI][Medline]
Iusem ND, Bartholomew DM, Hitz WD, Scolnik PA. (1993) Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiology 102 1353–1354.[CrossRef][ISI][Medline]
Kawasaki S, Miyake C, Kohci T, Fujii S, Uchida M, Yokota A. (2000) Response of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits. Plant Cell Physiology 41 864–873.
Khedr AHA, Abbas MA, Abdel Wahid AA, Quick WP, Abogadallah GM. (2003) Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. Journal of Experimental Botany 54 2553–2562.
Marrs KA. (1996) The functions and regulation of glutathione s-transferases in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47 127–158.[CrossRef][ISI][Medline]
Mittler R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7 405–410.[CrossRef][ISI][Medline]
Murota KI, Ohshita Y, Watanabe A, Aso S, Sato F, Yamada Y. (1994) Changes related to salt tolerance in thylakoid membranes of photoautotrophically cultured green tobacco cells. Plant Cell Physiology 35 107–113.
Neuhoff V, Arnold N, Taube D, Ehrhardat W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9 255–262.[CrossRef][ISI][Medline]
Noctor G, Veljovic-Jovanovic S, Driscoll S, Novitskaya L, Foyer CH. (2002) Drought and oxidative load in the leaves of C3plants: a predominant role for photorespiration? Annals of Botany 89 841–850.
Okuma E, Soeda K, Tada M, Murata Y. (2000) Exogenous proline mitigates the inhibition of growth of Nicotiana tabacum cultured cells under saline conditions. Soil Science and Plant Nutrition 46 257–263.
Pedrol N, Ramos P, Reigosa MJ. (2000) Phenotypic plasticity and acclimation to water deficits in velvet-grass: a long-term greenhouse experiment. Changes in leaf morphology, photosynthesis and stress-induced metabolites. Journal of Plant Physiology 157 383–393.
Riccardi F, Gazeau P, de Vienne D, Zivy M. (1998) Protein changes in response to progressive water deficit in maize: quantitative variations and identification. Plant Physiology 117 1263–1253.
Salekdeh GH, Siopongco HJ, Wade LJ, Ghareyazie B, Bennett J. (2002a) A proteomics approach to analysing drought- and salt-responsiveness in rice. Field Crops Research 76 199–219.[CrossRef]
Salekdeh GH, Siopongco HJ, Wade LJ, Ghareyazie B, Bennett J. (2002b) Proteomics analysis of rice leaves during drought stress and recovery. Proteomics 2 1131–1145.[CrossRef][ISI][Medline]
Samaras Y, Bressan RA, Csonka LN, Gracia-Rios MG, Paino D'Urzo M, Rhodes D. (1995) Proline accumulation during drought and salinity. In Smirnoff N (Ed.). Environment and plant metabolism: flexibility and accumulationOxford, UK BIOS Scientific Publishers pp. 161–187.
SAS Institute. (1996) SAS/STAT user's guide, Release 6.12 editionCary, NC.
Serrano R and Gaxiola R. (1994) Microbial models and stress tolerance in plants. Critical Reviews in Plant Science 13 121–138.
Silhavy D, Hutvagner G, Barta E, Banfalvi Z. (1995) Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense. Plant and Molecular Biology 27 587–595.
Smirnoff N. (1998) Plant resistance to environmental stress. Current Opinion in Biotechnology 9 214–219.[CrossRef][ISI][Medline]
Sugihara K, Hanagata N, Dubinsky Z, Baba S, Karube I. (2000) Molecular characterization of cDNA encoding oxygen-evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza. Plant Cell Physiology 41 1279–1285.
Wehmeyer N, Hernandez LD, Finkelstein RR, Vierling E. (1996) Synthesis of small heat-shock proteins is part of the developmental program of late seed maturation. Plant Physiology 112 747–757.[Abstract]
Yamaguchi-Shinozaki K, Kasuga M, Liu Q, Nakashima K, Sakuma Y, Abe H, Shinwari ZK, Seki M, Shinozaki K. (2002) Biological mechanisms of drought stress response JIRCAS Working Report.
Yokota A, Kawasaki S, Iwano M, Nakamura C, Miyake C, Akashi K. (2002) Citrulline and DRIP-1 protein (ArgE homologue) in drought tolerance of wild watermelon. Annals of Botany 89 825–832.
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