JXB Advance Access originally published online on March 30, 2006
Journal of Experimental Botany 2006 57(7):1537-1546; doi:10.1093/jxb/erj129
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RESEARCH PAPER |
Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology
Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466 Gatersleben, Germany
*To whom correspondence should be addressed. E-mail:mock{at}ipk-gatersleben.de
Received 22 June 2005; Accepted 23 January 2006
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Abstract |
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A proteome study based on 2-D gel electrophoresis was performed in order to analyse the cold-stress response of Arabidopsis plants. The emphasis was to monitor the overall changes in the protein complement after prolonged exposure rather than short-term responses. Two different temperature regimes were used (6 °C and 10 °C) and plants were exposed to cold-stress exposure for 1 week. Protein patterns were also monitored after re-shifting plants to control conditions for a further week. To monitor gradual changes in the response to the two cold-stress conditions, the analysis was performed with DIGE technology with the inclusion of an internal standard. In the experiments using 6 °C, 22 spots with at least 2-fold altered expression were found; among them 18 were increased and four were decreased. When plants were exposed to 10 °C, 18 of these 22 spots still showed a 2-fold change; however, the alterations were, in general, more moderate than observed under 6 °C. Spot identification was performed by MALDI-TOF and ESI-MS/MS. Many of the proteins identified have previously been described in the context of cold-stress responses, indicating the validity of this proteome approach for further in-depth studies.
Key words: 2-D DIGE, Arabidopsis, cold stress, differential in- gel electrophoresis, ESI-MS/MS, fluorescent dyes, MALDI-TOF, mass spectrometry, proteome analysis, two-dimensional gel electrophoresis
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Introduction |
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Plants have to cope with a range of biotic and abiotic stresses during their life cycle. Environmental cues such as high light, drought, high or low temperature, and salinity affect growth and yield of crop plants. Plants have developed a range of defence mechanisms in response to environmental stresses, among them a network of enzymes involved in the detoxification of excess reactive oxygen species which accumulate during an imbalance of cellular homeostasis. However, many other genes are involved in the cellular response to stresses, such as components of signalling networks. Characterization of various plant systems has revealed considerable overlap in the defence responses to different stresses on the transcript as well as on the level of enzyme activities (Apel and Hirt, 2004; Shinozaki et al., 2003). Further elucidation of the complex networks interacting during stress defences has recently been stimulated by the availability of techniques for the multi-parallel analysis of transcript levels in the model plant Arabidopsis and rice. The expression of 1300 Arabidopsis genes was monitored under cold and drought stress, and in a follow-up study, the expression patterns of 7000 genes was measured in response to cold, drought, and high salinity (Seki et al., 2001, 2002). The influence of these three abiotic stress factors on the transcriptome of rice was monitored using a microarray containing about 1700 genes (Rabbani et al., 2003). Expression of 8000 Arabidopsis genes was followed in an experiment involving cold, salt, and osmotic stress (Kreps et al., 2002). These and other studies have revealed a range of genes induced by one or several stresses, among them many transcription factors. The regulatory context of individual transcription factors within the network of cellular responses can then be studied in mutants and transgenic lines with modified expression of these genes, and by using comprehensive transcript profiling for the analysis of this plant material, target genes and pathways can be identified (Shinozaki et al., 2003).
Compared with the analysis of the transcriptome, analysis of the protein complement of plant tissues in response to cold or other abiotic stresses is still limited, but technical progress has been achieved within recent years in the separation of proteins and their sensitive identification by mass spectrometry (Canovas et al., 2004). A range of studies have demonstrated that the levels of transcripts and proteins are not strictly correlated as shown in yeast (Gygi et al., 1999; Ideker et al., 2001). In addition, many proteins are modified by post-translational modifications such as phosphorylation, glucosylation, ubiquitinylation, sumoylation, and many others (Mann and Jensen, 2001; Schweppe et al., 2003; Canovas et al., 2004). Many post-translational modifications are crucial for the regulation of protein function. Therefore, analysis of the cellular proteome complement is required in order to understand the cellular processes operating in response to environmental stresses. The most frequently used method to investigate differential protein abundance in large-scale proteomics experiments on crude protein mixtures is still two-dimensional gel electrophoresis (2-DE) despite limitations in separation capacity (O'Farrell, 1975; Görg et al., 2000; Rabilloud, 2002). A number of technical developments have been introduced to improve sensitivity, linearity, and reproducibility. The progress in 2-DE includes gels with narrower pH gradients, staining procedures with improved dyes based either on covalent or non-covalent labelling of proteins (Patton, 2000, 2002), but also more sophisticated software for the evaluation of gel patterns. A methodology to label proteins from pools to be compared with different dyes was originally introduced by Ünlü et al. (1997) and has been further developed and is now termed DIGE standing for differential in-gel electrophoresis (Tonge et al., 2001). The separate pools of proteins are covalently labelled with N-hydroxysuccinimidyl derivatives of the fluorescent cyanine dyes (Cy2, Cy3, Cy5). These fluorescent dyes are designed to modify the 
-amino group of lysine residues in proteins. Labelling of the proteins is limited by the quantity of dye added. By this means, approximately 3% of the available protein and one single lysine per protein molecule is labelled. The technique allows the analysis of up to three pools of protein samples simultaneously on a single 2-D gel, thereby minimizing the problem of gel-to-gel variability. In a standard protocol, two of the dyes (Cy3, Cy5) are used to label two protein samples to be compared, and the third dye (Cy2) is used to label an internal standard that consists of equal amounts of all the samples to be analysed within the overall experiment. The inclusion of such an internal standard allows experimental errors to be corrected and therefore improves the quantitative comparison of protein expression (Alban et al., 2003).
In the present manuscript, the 2-DE DIGE technique was used to monitor the alterations in the proteome of cold-stress-treated Arabidopsis plants. Cellular responses were investigated at two different temperature regimes and the experimental sep-up included a re-shift to control conditions. Proteins with at least 2-fold changes in abundance were selected for identification by mass spectrometry. By contrast with many short-term experiments used in transcriptome studies, the interest here was to elucidate the later adaptations on the protein level following initial signalling events.
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Materials and methods |
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Plant material, growth conditions and harvest
Arabidopsis thaliana (ecotype Col-0) seeds were germinated in a climate chamber under short-day conditions (9/15 h light/dark cycle, 20/18 °C day/night, 200 µmol m–2 s–1 light intensity). After cultivation for 2 weeks under these conditions 40 seedlings were transferred into trays on humus (Substrat 1, Klasmann–Deilmann GmbH, Geeste–Gross Hesepe, Germany) and maintained under the controlled conditions mentioned above in the climate chambers (Percival, CLF Laborgeräte GmbH, Emersacker, Germany). Plants were daily irrigated with water. Three weeks later half of the plants were shifted to the cold-stress conditions (6 °C or 10 °C, day and night) and the other half was used as a control (20/18 °C day/night). After exposure for 1 week under these conditions, stressed plants were shifted back to the control conditions and cultivated for an additional 7 d. After stress exposure and after re-shifting, rosette leaves were harvested, directly frozen in liquid nitrogen, and stored at –80 °C until further processing. Three completely independent sets of harvests (6 °C as well as 10 °C) were included in the proteome study.
Protein extraction
Plant material was ground in liquid nitrogen in a mortar to a fine powder. For protein extraction about 1 g (1 part) of this fine powder was mixed with 10 ml (10 parts) of precipitation solution containing 10% w/v TCA and 0.07% w/v 2-mercaptoethanol in acetone according to Damerval et al. (1986). Aliquots (1.8 ml each) of suspension were first chilled in liquid nitrogen for 15 s and then incubated at –20 °C for 45 min. After 5, 10, and 15 min the suspension was mixed. Precipitated material was collected by centrifugation (25 000 g, 4 °C, 15 min). After washing twice with acetone containing 0.07% w/v 2-mercaptoethanol the precipitate was dried in a vacuum centrifuge. Proteins were dissolved from the dried precipitate using 50 µl mg–1 of lysis-buffer (8 M urea, 2% CHAPS, 30 mM TRIS-HCl pH 8.5). No primary amines, DTT or ampholytes, were included in the lysis-buffer as such components could react with the NHS esters of the cyanine dyes. Insoluble material was pelleted by centrifugation (32 000 g, RT, 15 min) using a micro22R centrifuge (Hettich, Germany). The supernatant was additionally clarified by centrifugation through a 0.45 µm filter unit (ULTRAFREE-MC, Millipore, Eschborn, Germany). Protein concentration of these samples was determined by using the 2D-Quant-Kit (GE Healthcare/Amersham Biosciences, Freiburg, Germany) and BSA as standard.
Protein–cyanine dye labelling
Protein labelling was performed using the CyDyes DIGE Fluors developed for fluorescence 2-D DIGE technology (GE Healthcare/Amersham Biosciences) according to the manufacturer's recommended protocol. Each sample was covalently labelled with a different fluorophore, Cy2 (a mixture of equal amounts of protein extracts from control and treatment), Cy3 (control), and Cy5 (treatment). Cy2, Cy3, and Cy5 N-hydroxysuccinamide (NHS) esters were freshly dissolved in anhydrous N,N-dimethylformamide (DMF, 99.8%, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and centrifuged (12 000 g, RT, 5 min). In each case 50 µg of protein was labelled with 400 pmol of amine-reactive cyanine dyes. The labelling mixture was incubated on ice in the dark for 30 min and also centrifuged (12 000 g, RT, 5 min). The reaction was terminated by adding 1 µl of 10 mM lysine (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), which reacts with the remaining free NHS esters of the cyanine dyes, and incubated on ice for 10 min. Each of the labelled protein samples were mixed and rehydration buffer (8 M urea, 2% w/v CHAPS, 0.005% bromphenol blue) containing 20 mM DTT and 0.5% IPG buffer (GE Healthcare/Amersham Biosciences) were added to make up the volume to 250 µl prior to IEF.
2-D DIGE experimental design
To ensure statistical significance for data obtained from the control and cold-stress-treated plants, the following experimental design (Table 1) was applied. Equal amounts (50 µg) of control (Cy3), cold treatment (Cy5), and the mixture (Cy2) of the individually labelled samples were loaded per gel, resulting in 75 µg of protein from the control (20 °C) and each cold treatment (6 °C or 10 °C). For the assessment of biological variations, three gels from the independent sets of harvests (harvest 1–3) for each treatment were performed. In addition, three gels for each treatment were performed for harvest 1 to assess experimental variation. A total of 24 gels (12 for biological variation (6 °C and 10 °C) and 12 for technical repetition) were run to evaluate statistically the protein expression variation between the control and the cold-treatment conditions.
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2-D gel electrophoresis
CyDyes labelled samples (150 µg protein in total) were loaded by rehydration to strips of 13 cm in length with immobilized pH gradient of 4–7 and separated on an IPGPhor Unit (GE Healthcare/Amersham Biosciences), using the following settings: 1 h 250 V, 1 h 500 V, 1 h gradient from 500 to 4000 V, 5 h 4000 V for a total of about 24 kVh. After IEF, strips were equilibrated for 15 min in equilibration buffer (50 mM TRIS–HCl, pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 20 mM DTT, 0.01% w/v bromphenol blue) and mounted on top of a 11.25% SDS–polyacrylamide gel with stacking gel (Laemmli, 1970) in a Hoefer S600 apparatus (GE Healthcare/Amersham Biosciences). Electrophoresis was performed at 150 V constant voltage.
Gel imaging and data analysis
After SDS–PAGE, cyanine dye-labelled proteins were visualized directly by scanning using a TyphoonTM 9400 imager (GE Healthcare/Amersham Biosciences). Cy2 images were scanned using a 488 nm laser and an emission filter of 520 nm band pass (BP) 40. Cy3 images were scanned using a 532 nm laser and a 580 nm BP 30 emission filter. Cy5 images were scanned using a 633 nm laser and a 670 nm BP 30 emission filter. All gels were scanned at 100 µm (pixel size) resolution. The photomultiplier tube (PMT) was set to 500 V by using normal sensitivity. The scanned gels were than directly transferred to the ImageQuant V5.2 software package (GE Healthcare/Amersham Biosciences). Gel images were converted to 16-bit TIF files and were processed using the DeCyder software V4.0 (GE Healthcare/Amersham Biosciences). Each individual Cy3 gel image was assigned as control (20 °C) and all Cy5 images were assigned as treatment (6 °C as well as 10 °C). All Cy2 results were defined as standards when comparing gels from control and treatment.
After image analysis including the Student's t-test, spots of interest were picked. For picking spots parallel gels loading 100 µg protein were run and stained with colloidal Coomassie G 250 (GelCode Blue Stain Reagent, Pierce 24592, Rockford, USA).
Identification of proteins by mass spectrometry
Gel pieces were washed for 30 min at room temperature with vigorous shaking in 400 µl buffer (10 mM ammonium bicarbonate/50% acetonitrile). After removing the supernatant, the gel pieces were dried. For the digestion of protein spots, 10 µl trypsin solution (Sequencing Grade Modified Trypsin V511, Promega, Madison; 10 ng µl–1 in 5 mM ammonium bicarbonate/5% acetonitrile) were added to each sample. After incubation for 5 h at 37 °C, the reaction was stopped by adding 2 µl 1% TFA.
For MALDI-TOF mass spectrometry, 1 µl of the digest was mixed with 2 µl of the matrix solution (5 mg 
-cyano-4-hydroxycinnamic-acid in 80% v/v acetonitrile and 0.1% w/v TFA) and 1 µl of this mixture was deposited onto the MALDI target. Tryptic peptides were analysed with a REFLEX III MALDI-TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany). Spectra were calibrated using trypsin autolysis products (m/z 842.50+ and 211.10+) as internal standards under application of the XMASS software Version 5.1.5 (Bruker Daltonics, Bremen, Germany).
Protein identification was performed by searching for Viridiplantae in the non-redundant NCBI database using the Mascot search engine (Matrix Science; London, UK) using the following parameters: monoisotopic mass accuracy, 100 ppm; missed cleavages, 1; allowed variable modifications, oxidation (Met) and propionamide (Cys).
For the ESI and the de novo sequencing experiments, 3 µl of the digest was subjected to nanoscale LC-ESI MS/MS analysis. The nanoscale RP LC analyses were conducted on a nanoAcquity UPLC system (Waters Corporation, Milford, MA, USA). The mobile phase flow from the binary pump was used to preconcentrate and desalt the digest samples on a 20 mm x180 µm Symmetry 5 µm C18 precolumn (Waters Corporation) for 3 min at 4 µl min–1 with an aqueous 0.1% formic acid solution. The peptides were subsequently eluted onto a 10 mm x75 µm analytical Atlantis C18 column (Waters Corporation) and separated at 0.3 µl min–1 with an increasing ACN gradient from 2% to 40% B in 30 min. The mobile phase A consisted of 0.1% formic acid in water and the mobile phase B of 0.1% formic acid in ACN. The nanoscale LC effluent from the analytical column was directed to the NanoLockSpray source of a Q/Tof Premier hybrid orthogonal accelerated Time-of-Flight (oa-ToF) mass spectrometer (Waters Corporation, MS Technologies Centre, Manchester, UK). The mass spectrometer was operated in a positive ion mode with a source temperature of 80 °C and a cone gas flow of 30 l h–1. A voltage of approximately 2 kV was applied to the nano flow probe tip. The mass spectra were acquired with the TOF mass analyser in the V-mode of operation and spectra were integrated over 1 s intervals. MS and MS/MS data were acquired in a continuum mode using MassLynx 4.0 software (Waters Corporation, Technologies Centre). The instrument was calibrated with a multi-point calibration using selected fragment ions of the CID of Glu-Fibrinopeptide B (SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany). Automatic data directed analysis was employed for MS/MS analysis on doubly and triply charged precursor ions. The product ion MS/MS spectra were collected from m/z 50 to m/z 1600. Lock mass correction of the precursor and the product ions was conducted with 150 pmol µl–1 Glu-Fibrinopeptide B in 0.1% formic acid in ACN/water (25:75, v/v) respectively, and introduced via the reference sprayer of the NanoLockSpray interface. ProteinLynx GlobalSERVER v2.1 software was used as a software platform for data processing, deconvolution, and de novo sequence annotation of the spectra, and various database search types. The MS/MS spectra searches were conducted with a protein Viridiplantae index of the non-redundant NCBI database. A 10 ppm peptide, 0.1 Da fragment tolerance, one missed cleavage, and variable oxidation (Met) and propionamide (Cys) were used as the search parameters. BLAST homology and similarity searches were conducted with a protein Viridiplantae index of the non-redundant NCBI database.
Statistical analysis
Results from three different experiments were compared for control (20 °C) and cold-stress treatment (6 °C and 10 °C) and differences in spot abundance statistically evaluated using the t-test function implemented into the DeCyder software for the analysis of the 2-DDIGE gels. Means and standard deviations were calculated from three independent sets of harvests (biological repetition) as well as from three iterations of one harvest (technical replication) and compared between control and treatment. The number of detected spots showing differences with a P-value of 
0.05 was then determined.
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Results |
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General description of the experiments
Arabidopsis thaliana plants were cultivated under different temperature regimes, 6 °C and 10 °C as the stress condition and 20 °C for control as described in the Materials and methods. The experimental design is illustrated in Fig. 1A. Cultivation of Arabidopsis plants under cold-stress conditions leads to reduced growth at both treatments used [Fig. 1B (b) and (c)] relative to the control plants [Fig. 1B (a)]. The delay in growth was also visible 1 week after the re-shift to control conditions but not as pronounced as directly after the cold-stress treatments [Fig. 1B (d–e)]. Beside the reduced plant size no obvious morphological phenotype was observed under the growth conditions used.
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Diverse 2-D DIGE protein patterns of control and cold-stress-treated plants
To detect proteins variably expressed in the cold-stress treatment, 2-D DIGE patterns from control and the corresponding stress treatment were compared. Extracts were prepared from rosette leaves of Arabidopsis thaliana control and treated plants and separated by 2-D gel electrophoresis as described in the Materials and methods, also outlined in Table 1. In Fig. 2A a representative image from a 2-D DIGE gel of the 6 °C treatment is shown. Selected channels are corresponding to the control (Cy3, Fig. 2B), stress treatment (Cy5, Fig. 2C), and the mixed sample (Cy2, Fig. 2D), respectively.
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The comparative image analysis revealed 22 spots, which showed at least 2-fold different protein expression by comparing samples from cold stress (6 °C) and control treatment (Fig. 3). Eighteen of these spots were increased and four protein spots (spot number 19–22) showed decreased accumulation in the stress-treated plants (6 °C) when compared with the controls (Table 2). Effects of the cold treatment on the protein patterns were diminished at 10 °C relative to the 6 °C treatment, but still 16 of these proteins were up- and two were down-regulated. For the high accumulating proteins (spots 8, 13, 14, and 15) the increase in spot volumes were lower for the plants grown at 10 °C stress treatment relative to the patterns observed in the 6 °C treatment (Table 2).
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2-D DIGE protein patterns of control and cold-stress-treated plants after re-shift to control conditions
To characterize the cellular reactions in plants after recovery from cold stress, the protein patterns of Arabidopsis rosette leaves of plants further cultivated for 1 week under control conditions were investigated upon stress exposure. For most of the spots, protein accumulation shifted back to the level of the control plants. An example is given for a group of proteins in Fig. 4. In the magnified areas increased expression values under cold treatment (C–E) and similar expression values of these proteins relative to control after the re-shift experiment (F–H) is illustrated. This re-shift was not observed for spot one which shows still higher accumulation at both treatments relative to the control 7 d after the change back to control conditions (Table 2). Still higher but not 2-fold increased protein accumulation in the treated plants compared with control plants was observed 7 d after the re-shift for spots 5, 10, and 18. Furthermore, down-regulated protein values shifted back to the level of the control in the 10 °C-treated plants; a tendency also observed in the 6 °C-treated plants (Table 2).
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For all different expressed proteins in both cold-stress treatments as well as for the shift and the re-shift experiments, similar protein expression levels were observed in the biological variation experiment (harvests 1–3) and in the technical repetition of experiment one. As an example, magnification of spot number 17 from the original gels is shown in Fig. 5 for both experimental set-ups. A more than 2-fold increased protein spot volume was observed in the 6 °C as well as in the 10 °C treatment for both experimental designs (Fig. 5A, B (Cy5); Table 2). Furthermore, comparison of the normalized spot volume revealed nearly equal values for the different harvests as well as for the technical replications (data not shown).
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Spot identification by mass spectrometry
The 22 spots (Fig. 3) showing at least a 2-fold change in protein expression were chosen and digested as described in the Materials and methods. Many of the proteins found to exhibit different accumulation patterns after cold-stress treatment could be identified using MALDI-TOF-MS. Peptide mass fingerprinting was also used to confirm the identity of the annotated spots from independent gels. The proteins identified include a putative RNA-binding protein, a glycine-rich protein, 60S ribosomal protein P2, a putative major latex protein, Rieske protein (petC), a putative S-adenosylmethionine synthase, a putative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, a dehydrin (ERD 10), a low-temperature-induced protein 78, and a ß-1,3-glucanase 2 from Arabidopsis thaliana (Table 3). However, for some of the low abundant spots, the information was not sufficient for identification. For those samples de novo sequencing allowing searching databases for homologue sequences from related plant species was performed. Database searches revealed homology for a peptide of spot 20 with stretches of a hypothetical protein from Arabidopsis thaliana (Table 3). For spot 16, peptide sequences with homology to two different Arabidopsis proteins have been revealed from database searches (data not shown) which have to be clarified in further analyses.
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Discussion |
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In the present study, cold-stress-induced changes in the proteome of Arabidopsis thaliana rosette leaves were analysed after exposure for 7 d. Plants kept under 6 °C showed 22 spots with at least a 2-fold change in protein abundance, whereas in plants exposed to 10 °C 18 spots were monitored. For most of these 18 spots, the changes in protein abundance were more pronounced under the lower temperature, as summarized in Table 2. Among the 22 spots with at least a 2-fold change at 6 °C, 18 were up- and four were down-regulated.
Out of these 22 proteins, 13 were identified by MALDI-TOF alone or also by ESI-MS/MS; one spot could only be identified by ESI-MS/MS.
Many of the spots identified in these experiments have already been discussed in the context of cold-stress responses. RNA-binding protein CP29 (spot 2) has been shown to be localized in chloroplasts. In maize, cold-resistant and -sensitive lines differed in the phosphorylation of CP29, which is a subunit of the photosystem II. In the non-phosphorylating genotype, cold stress was followed by photo-inhibitory damage (Mauro et al., 1997). A glycine-rich protein (spot 3) was shown to increase the freezing tolerance of Arabidopsis plants (Kim et al., 2005).
Three out of the 22 spots were identified as dehydrin (ERD 10; At1g20450); they might represent modifications of this protein or closely related proteins of this family, which could not be differentiated by mass spectrometry. Dehydrins, assigned as the D-11 family of late embryogenesis abundant (LEA) proteins, are formed during stresses having dehydration as a component, such as salinity, drought, and low temperature. They are believed to function as stabilizers having detergent and chaperone-like properties (Close, 1996, 1997). Induction of dehydrins by cold stress has been reported for rice (Lee et al., 2005), wheat (Ohno et al., 2003), in fruits of Citrus species (Porat et al., 2004), as well as for Arabidopsis (Nylander et al., 2001; Kawamura and Uemura, 2003).
As indicated by name, a low-temperature-induced protein 78 (spot 17) has also been found to be induced by cold stress (Nordin et al., 1993).
Several of the proteins identified in these experiments were also identified in transcriptome studies on cold-stress responses, such as the glycine-rich protein (spot 3), dehydrins (spots 13–15), and the low-temperature-induced protein 78 (spot number 17). However, different kinetics for transcript and protein levels have to be considered, and the harvest for the transcript profiling was performed at much earlier time points than in this study's experimental set-up (Kreps et al., 2002).
These results demonstrate that, with a proteome approach, a comprehensive set of proteins associated with cellular responses to cold stress can be detected. Proteome analysis therefore allows the global changes associated with the adaptations to stresses within the protein complement of plants to be monitored. These experiments indicate that by applying an internal standard such as within the DIGE technology, the influence of gradual changes in the stress conditions on the abundance of stress-associated proteins can be monitored.
However, extension of the experimental approach is needed in order to cover a wider part of the proteome, as a prerequisite for an in-depth understanding of the regulatory circuits at the level of proteins comparable to the resolution already reached in transcriptome analysis of stress defence responses. Such approaches will include the use of narrower pH gradients and pre-fractionation techniques to analyse sub-proteomes. Parallel to the use of 2-DE based separation, chromatographic techniques will also add valuable information.
In conclusion, these results demonstrate the capacity of proteome approaches to analyse the cellular mechanisms at the level of proteins in order to understand the complexity of plant defence responses, despite current technical limitations.
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Acknowledgements |
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Funding by the DFG with grants to H-P Mock (Mo 479/4-1 and 4-2) is gratefully acknowledged. We wish to thank P Linow and A Wolf for their excellent technical assistance, E Geyer and the greenhouse personnel for taking care of the plants and H Ernst for photographic work. The authors thank D Markowsky (GE Healthcare/Amersham Bioscience Company) for his help in the evaluation of the DIGE analysis.
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
, , , , , , . . . , –.A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics (2003) 3:36–44.[CrossRef][Web of Science][Medline]
, . . . , –.Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology (2004) 55:373–399.[CrossRef][Medline]
, , , , , . . . , –.Plant proteome analysis. Proteomics (2004) 4:285–298.[CrossRef][Web of Science][Medline]
. . . , –.Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum (1996) 97:795–803.[CrossRef]
. . . , –.Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiologia Plantarum (1997) 100:291–296.[CrossRef]
, , , . . . , –.Technical improvments in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis (1986) 7:52–54.
, , , . . . , –.Correlation between protein and mRNA abundance in yeast. Molecular and Cellular Biology (1999) 19:1720–1730.
, , , , , , . . . , –.The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis (2000) 21:1037–1053.[CrossRef][Web of Science][Medline]
, , , , , , , , , . . . , –.Integrated genomic and proteomic analysis of a systematically perturbed metabolic network. Science (2001) 292:929–934.
, . . . , –.Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. The Plant Journal (2003) 36:141–154.[CrossRef][Web of Science][Medline]
, , . . . , –.Cold-inducible zinc finger-containing glycine-rich RNA-binding protein contributes to the enhancement of freezing tolerance in Arabidopsis thaliana. The Plant Journal (2005) 42:890–900.[CrossRef][Web of Science][Medline]
, , , , , . . . , –.Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiology (2002) 130:2129–2141.
. . . , –.Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
, , , , , . . . , –.Characterisation of an abiotic stress-inducible dehydrin gene, OsDhn1, in rice (Oryza sativa L.). Molecules and Cells (2005) 19:212–218.[Medline]
, . . . , –.Proteomic analysis of post-translational modifications. Nature Biotechnology (2001) 21:255–261.
, , , . . . , –.Cold-resistant and cold-sensitive maize lines differ in the phosphorylation of the photosystem II subunit, CP29. Plant Physiology (1997) 115:171–180.[Abstract]
, , . . . , –.Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh. Plant Molecular Biology (1993) 21:641–653.[CrossRef][Web of Science][Medline]
, , , . . . , –.Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Molecular Biology (2001) 45:263–279.[CrossRef][Web of Science][Medline]
. . . , –.High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry (1975) 250:4007–4021.
, , . . . , –.Kinetics of transcript and protein accumulation of a low-molecular-weight wheat LEA D-11 dehydrin in response to low temperature. Journal of Plant Physiology (2003) 160:193–200.[CrossRef][Web of Science][Medline]
. . . , –.A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis (2000) 21:1123–1144.[CrossRef][Web of Science][Medline]
. . . , –.Detection technologies in proteome analysis. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences (2002) 771:3–31.
, , , , , , , . . . , –.Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that specially induced by the combination of heat followed by chilling temperatures. Physiologia Plantarum (2004) 120:256–264.[CrossRef][Medline]
, , , , , , , , , . . . , –.Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and absinic acid application using cDNA microarray and RNA gel-blot analysis. Plant Physiology (2003) 133:1755–1767.
. . . , –.Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics (2002) 2:3–10.[CrossRef][Web of Science][Medline]
, , , , . . . , –.The characterisation of protein post-translational modifications by mass spectrometry. Accounts of Chemical Research (2003) 36:453–461.[CrossRef][Web of Science][Medline]
, , , , , , , . . . , –.Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. The Plant Cell (2001) 13:61–72.
, , , et al. . . , –.Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal (2002) 31:279–292.[CrossRef][Web of Science][Medline]
, , . . . , –.Regulatory network of gene expression in the drought and cold responses. Current Opinion in Plant Biology (2003) 6:410–417.[CrossRef][Web of Science][Medline]
, , , , , , , , , . . . , –.Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics (2001) 1:377–396.[CrossRef][Web of Science][Medline]
, , . . . , –.Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis (1997) 18:2071–2077.[CrossRef][Web of Science][Medline]
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