JXB Advance Access originally published online on March 17, 2006
Journal of Experimental Botany 2006 57(7):1553-1562; doi:10.1093/jxb/erj149
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RESEARCH PAPER |
Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures
School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK
*To whom correspondence should be addressed. E-mail: a.r.slabas{at}durham.ac.uk
Received 10 April 2005; Accepted 6 February 2006
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionConclusionReferences |
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Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen–host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization–time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.
Key words: Arabidopsis, 2-D DIGE, defence response, fungal elicitor, molecular chaperones, oxidative stress, plant pathogen, proteomic
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plants continuously encounter microorganisms at almost every stage of their development. However, only a minority of these encounters result in their tissues being invaded by microbes causing deleterious effects, such as diversion of nutrients and essential metabolites, and toxin production that culminates in the development of disease and sometimes death. Consequently, the interaction between plants and pathogens is of immense commercial importance as it can lead to massive yield penalties, and so an understanding of the mechanisms by which plants resist infection or mount a defensive response is vital from an agricultural standpoint.
A combination of biochemical and genetic approaches has established that the interaction of plants and microorganisms is complex. Plants mount a defensive response to some pathogens after perception of pathogen presence via the physical interaction of pathogen-derived elicitor molecules with the cognate plant receptors (Leister et al., 1996; Dangl and Jones, 2001; Nürnberger and Brunner, 2002). In some cases, this interaction occurs between certain plant cultivars and specific pathogen races and is genetically determined by complementary pairs of pathogen-encoded avirulence genes and plant resistance genes. In this gene-for-gene interaction (Flor, 1971), mutation of either the avirulence gene or the resistance gene leads to the failure by the plant to recognize the pathogen and the pathogen successfully colonizes the host and disease ensues. However, induced resistance by plants to some pathogens is not based on the gene-for-gene interaction, but still involves surveillance systems of receptor molecules that can recognize diverse elicitor molecules of pathogen origin and is functional against a wide spectrum of pathogens (Nürnberger et al., 2004). Regardless of whether the elicitor is race-specific or a general elicitor, the downstream events that the elicitor-receptor binding triggers are the same.
Following this initial recognition, a series of biochemical events is triggered and includes a Ca2+ influx (Stab and Ebel, 1987; Bach et al., 1993), alkalinization of the extracellular matrix (Felix et al., 1993), and a burst of active oxygen species (Chai and Doke, 1987; Legendre et al., 1993). There is evidence that all of these early events are needed for the successful inauguration of a robust defence response (Stab and Ebel, 1987; Jabs et al., 1997; Bolwell et al., 1995, 1999). A highly localized, rapid, programmed cell death known as the hypersensitive reaction (Lam et al., 2001) is triggered downstream of these biochemical changes. This appears to function in defence by isolating the pathogen and depriving it of essential nutrients, and impeding its spread if it requires living cells as a conduit for movement. Several changes in the structure of the cell wall are instigated during the early stages of this defence response. Proline-rich and hydroxyproline-rich glycoproteins are cross-linked to the cell wall (Bradley et al., 1992; Brisson et al., 1994), resulting in the reinforcement of the cell wall, a physical barrier to pathogen ingress. The resultant fortified cell walls become less vulnerable to digestion by microbial enzymes (Brisson et al., 1994).
Biosynthesis and accumulation of an array of endogenous signalling compounds like H2O2 (Bolwell et al., 2002), nitric oxide (Delledone et al., 1998), salicylic acid (Malamy et al., 1990), ethylene (Penninckx et al., 1998), and jasmonic acid (Penninckx et al., 1998; Schmelz et al., 2003) follows the initial activation of the defence response. These secondary signals in turn set in motion signal transduction cascades that eventually lead to activation of pathogen-responsive genes. The nature of the genes switched on during the pathogen–plant host interaction is subject to intense interest as some of the products of these genes are directly responsible for fending off the pathogen attack and rendering the plant immune to subsequent attacks. A subset of these genes is likely to be key signalling components mediating the development of this systemic acquired resistance.
The ultimate events in the establishment of the pathogen- or elicitor-induced resistance is the local and systemic synthesis of antimicrobial secondary metabolites like phytoalexins (Kuc and Rush, 1985) and synthesis and accumulation of defence-related proteins including pathogenesis-related (PR) proteins. Several of the PR proteins have hydrolytic activities against fungal and bacterial pathogens. For example, PR-2 family members have 1,3-ß-glucanase activity while PR-3, PR-4, PR-8, and PR-11 families have chitinase activity (Fritig et al., 1998). Although the enzymatic activities of PR-1 and PR-5 are unknown, they have antifungal activity in vitro via a mechanism that leads to perforation of fungal membranes (Niderman et al., 1995; Abad et al., 1996).
Some of the signalling components in the induction of defence-related proteins have been identified. For example, the product of an Arabidopsis gene (PDF1.2) differentially expressed in response to attack by Alternaria brassicicola was identified as a defensin with antifungal properties in vitro (Penninckx et al., 1996). Mutant analyses revealed that EIN2 and COI1, components of ethylene and jasmonate signalling, respectively, are key factors upstream of PDF1.2 induction and systemic acquired resistance to fungal pathogens (Penninckx et al., 1998; Thomma et al., 1999). Studies of this nature have successfully dissected out some of the components involved in the induction of individual defence proteins. However, to gain complete understanding of pathogen defence systems, there is a need to identify all the diverse signalling cascades of multiple biochemical pathways activated by the single elicitation event and working synergistically to establish a heightened resistance status, the hallmark of systemic acquired resistance. With the recent advances in technologies allowing large-scale monitoring of thousands of genes or gene products in a single experiment, there has been considerable progress in identifying global changes in gene expression during pathogen attack.
A number of such studies utilizing DNA microarrays or DNA chips has been carried out to investigate the global changes in the transcriptome induced by pathogens in Arabidopsis (Schenk et al., 2000; Ramonell et al., 2002; Maleck et al., 2004; Zimmerli et al., 2004). However, transcriptional changes do not reflect the complete cellular regulatory processes, since post-transcriptional processes, altering the amount of active protein, such as synthesis, degradation, processing, and modification of proteins, are not taken into account. Thus, complementary approaches such as proteome-based expression profiling are needed to obtain a full picture of the regulatory elements in plant–pathogen interactions.
In this study, the model organism Arabidopsis thaliana has been used to identify differentially expressed proteins in response to treatments with pathogen-derived elicitors. A cell suspension culture system was used that has the advantage of having uniform cells and, therefore, ensures greater reproducibility of the biological response. Moreover, two-dimensional difference gel electrophoresis (2-D DIGE) was used (Ünlü et al., 1997; Tonge et al., 2001) for identifying responsive protein spots, a method that greatly reduces the effects of gel-to-gel variation due to the incorporation of an internal pooled standard across all gels.
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Materials and methods |
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Cell culture treatments and protein extraction
Arabidopsis cell suspension cultures were grown in the dark for 3 d and aliquots were treated with water (to serve as controls) or a final concentration of 400 µg ml–1 Fusarium elicitor in a final volume of 5 ml. The elicitor was prepared as described before (Raventos et al., 1995). After 24 h of incubation, the cells were harvested by filtering through two layers of Miracloth and resuspended in 300 µl of 10% trichloroacetic acid. The acidified cells were snap-frozen in liquid nitrogen and stored at –20 °C. The cells were thawed and homogenized with plastic micropestles in the presence of 100 mg of sand. The homogenates were centrifuged for 10 min at 16 000 g. The supernatants were discarded and the pellets washed three times with 500 µl 80% acetone by repeated resuspension and centrifugation. The final pellets were washed once with the same volume of absolute acetone and dried by briefly blowing compressed air over them. Precipitated protein was extracted by resuspending the pellets in 500 µl of a urea buffer (9 M urea, 2 M thiourea, 4% CHAPS) and incubating at room temperature on an orbital shaker (180 rpm) for at least 30 min. Insoluble material was removed by centrifuging for 10 min at 16 000 g and discarding the pellets.
Sample clean-up and fluorescent labelling
Protein aliquots of 100 µl each from these preparations were stripped of non-protein contaminants using a GE Healthcare (Amersham, UK) 2-D Clean-Up Kit following the manufacturer's instructions. The cleaned protein was resolubilized in a TRIS-buffered solution (9 M urea, 2 M thiourea, 4% CHAPS, 30 mM TRIS-Cl pH 9) and adjusted to pH 8.5 using NaOH. Protein concentration was determined by a modified Bradford assay (Ramagli and Rodriguez, 1985) against a bovine serum albumin standard.
Stock solutions of CyDye DIGE Fluor (GE Healthcare, UK) were prepared by reconstituting the lyophilized powder in DMF (dimethylformamide) to a final concentration of 1 nmol µl–1. The stock was further diluted 2 in 5 using DMF to give a working solution of 400 pmol µl–1. Sample aliquots containing 50 µg protein in 18 µl buffer (9 M urea, 2 M thiourea, 4% CHAPS, 30 mM TRIS-Cl pH 8.5) were spiked with 1 µl of the CyDye working solution and incubated for 30 min on ice in the dark. The labelling reaction was terminated by the addition of 1 µl of 10 mM lysine. For labelling protein amounts over 50 µg, the volumes of all solutions were scaled up proportionally to maintain the same ratio of 400 pmol CyDye to 50 µg protein in 20 µl final reaction volume. Since six independent experiments were conducted, there were 12 protein samples in total. Control samples were labelled with CyDye DIGE Fluor Cy3, while elicitor-treated samples were labelled with CyDye DIGE Fluor Cy5. A pooled standard containing 25 µg protein from each of the 12 samples was labelled with CyDye DIGE Fluor Cy2.
Two-dimensional gel electrophoresis and image acquisition
Protein mixtures for a single gel were prepared by mixing aliquots with 12.5 µg labelled protein each of the Cy2-labelled pooled standard, Cy3-labelled control, and Cy5-labelled elicitor-treated samples. The volume of the mixtures was made up to 70 µl to give a final concentration of 9 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 2% IPG buffer pH 4–7. Immobiline DryStrips (18 cm pH 4–7 linear; GE Healthcare) were rehydrated overnight using sample buffer (9 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 2% IPG buffer pH 4–7). The protein mixtures were loaded into the rehydrated first-dimension gels using the cup-loading technique. Isoelectric focusing was performed using the Ettan IPGphor (GE Healthcare). During isoelectric focusing, the temperature was kept at 25 °C and a maximum current of 50 µA per gel was set. A total focusing of 70 kVh was achieved by following a running protocol with four phases of stepped voltages from 500 to 6500 V. Prior to the second dimension, the gels were equilibrated, reduced, and alkylated as described previously (Chivasa et al., 2002). The proteins were separated in 12% polyacrylamide second-dimension gels using the Ettan DALT Twelve System (GE Healthcare). These analytical gels were initially run at 5 W per gel for 30 min and subsequently at 17 W per gel until the bromophenol blue reached the bottom of the gels.
Gel images were acquired by scanning the six gels with the Typhoon 9400 variable mode imager (GE Healthcare). Cy2 images were scanned using a blue laser (488 nm) at an emission wavelength of 520/40 nm (maxima/band width). A green laser (532 nm) was used to scan Cy3 images at an emission wavelength of 580/30 nm. Cy5 images were acquired after excitation with a red laser (633 nm) using an emission filter of 670/30 nm. All gels were scanned at a resolution of 100 µm using PMT voltages that did not allow saturation of the most intense spot on each image.
Gel analysis
The DeCyderTM Differential Analysis Software Version 5.00 (GE Healthcare) was used for gel analysis. Triplicate gel images consisting of the Cy2-labelled pooled standard and the respective Cy3 and Cy5 samples in each gel were processed using the DeCyder DIA (Differential In-gel Analysis) software to co-detect and quantify the protein spots in the images. The spot detection algorithm automatically merges the three images and incorporates all the spot features into a single virtual image. After spot detection, which utilizes the pixel data from the three raw images and the merged image, values (volume, area, height, slope, etc.) for individual spots are calculated. The ratio of spot volume between the Cy3 and Cy5 images is calculated for each spot and this indicates the change in spot volume between the two samples. The algorithm then normalizes the ratio values so that the modal peak of volume ratios is zero. This DIA co-detection, quantification, and normalization process was performed automatically for all gels using the DeCyder batch processor. The estimated number of spots for each co-detection was set at 3500. Matching across gels was performed using the DeCyder biological variation analysis module to allow for statistical analysis of changes in protein abundance between the control and elicitor-treated samples. Paired t tests were performed for each protein spot and only proteins positively identified by mass spectrometry and showing a significant (P <0.05) quantitative change (over 20%) in response to the elicitor are reported.
Preparative gels and protein identification
Aliquots with 200 µg and 400 µg of unlabelled protein from control and elicitor-treated samples were separated by 2-D electrophoresis as described above. These preparative gels were fixed in a solution containing 40% (v/v) methanol and 10% (v/v) glacial acetic acid. After 12 h, the gels were incubated with fresh fixing solution for another 12 h and then gels with 200 µg protein were stained overnight with SyproTM Ruby solution in the dark (Genomic Solutions, Huntingdon, UK). These gels were destained for 4 h by incubating with 10% (v/v) methanol:6% (v/v) acetic acid. Imaging was performed using the Typhoon 9400 at an excitation wavelength of 532 nm and 610/30 nm emission filter. The images were matched back to DIGE analytical gels using DeCyder software and a picking list of proteins of interest was generated. The same gels were re-imaged on a ProPick Workstation (Genomic Solutions) and the protein spots of interest excised from the gels for processing by mass spectrometry. Proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as described previously (Simon et al., 2002). The peptide masses generated via MALDI-TOF were used to search the NCBI database, found at http://www.ncbi.nlm.nih.gov, and the Mowse score cut-off point for a positive identification was 64.
Histochemical localization of H2O2 and pH measurements
Cell cultures were treated with water (controls) or elicitor and the pH of the medium was measured every 20 min for 1 h. Accumulation of H2O2 in cell cultures was visualized by histochemical staining with 3,3'-diaminobenzidine (DAB). Cell cultures were spiked with 10 µg ml–1 DAB and treated with elicitor in the presence or absence of 10 mM ascorbic acid. After 6 h, a lawn of cells from each treatment was photographed in multi-well plastic plates.
RNA analysis
Total RNA was extracted from 100 mg of cells using an RNeasy Plant Mini kit (Qiagen, Crawley, UK) with on-column DNase digestion, according to the manufacturer's instructions. RNA was eluted in RNase-free sterile water. First-strand cDNA was synthesized with 5 µg total RNA using 200 units of SuperScript III reverse transcriptase (Invitrogen, Paisley, UK) and 500 ng oligo (dT)15 (Promega, Southampton, UK) in a final volume of 20 µl. The reaction mixture was incubated at 50 °C for 2 h and terminated with 15 min incubation at 70 °C.
Amplification products were quantified in real-time reverse transcription-PCRs using SYBR Green I to monitor dsDNA synthesis. The reaction proceeded in a 20 µl volume containing 0.25 mM dNTPs, 0.2 µM forward and reverse primers, and 1.5 µl of a 1:4 dilution of cDNA per reaction. SYBR Green I (Molecular Probes, Leiden, The Netherlands) was used at a final dilution of 1:60 000, using sterile nuclease-free water to make the stock dilution. Cycling conditions used were as follows: denaturation at 95 °C for 2 min, followed by 40 cycles at 95 °C for 20 s, annealing at 56 °C for 20 s, and extension at 72 °C for 40 s. Thermal cycling occurred in a Rotor-Gene 3000 (Corbett Research, Sydney, Australia). Oligonucleotide primers designed to amplify approximately 250–300 bp products from cDNA were synthesized by Sigma-Genosys (Haverhill, UK). The following primer pairs were used in PCRs to amplify the quinone reductase (At5g54500), 5'-TCAGTTCAAAGCCTTTTTGGATGC-3' and 5'-CTAAG-CAGTAGATCCCTTGAGCTTC-3'; glutathione S-transferase (At1g02920), 5'-TTGTTTGGGAGC-AAGTCTTAAAGC-3' and 5'-TTAAAGAACCTTCTTAGCAGAAGGCC-3'; mitochondrial aldehyde dehydrogenase (At3g48000), 5'-TCGCTCAAGACGAGATTTTCGGTCC-3' and 5'-TCAG-ATCCAGGCAGGCTTATTTAGAGC-3'; vacuolar ATP synthase subunit B (At4g38510), 5'-AGGACGT-GCAGGCCATGAAAGC-3' and 5'-TCAGTTGGTGGTATCGCGACTG-3'; and thioredoxin (At4g04950), 5'-TCAGCTTTACGTGAAAGGCGAGCT-3' and 5'-TCGGATAGAGTTGCTTTGAGATCACC-3'.
Control reactions lacking either first-strand cDNA or oligonucleotide primers, or containing only total RNA which had not been reverse transcribed, did not yield amplification products. PCR products were resolved on 0.8% agarose gels to confirm the presence of a single DNA band at the expected size. Fluorescence was detected at 510 nm with excitation at 470 nm at 72 °C. Melt curve analysis was used to verify the presence of specific DNA products with high melting temperatures as follows: samples were held at 50 °C for 30 s, rising by 1 °C s–1 up to 99 °C whilst monitoring the fluorescence (F). Amplificant melting temperatures were indicated by peaks in dF/dT plots. Quantification of cDNA abundance was achieved by comparative quantitation calculations using Rotor-Gene software. Samples were normalized only by reference to the quantity of total RNA used in the reverse transcription-PCRs (
94 ng). However, both thioredoxin and vacuolar ATP synthase subunit B2 transcript abundance remained at fairly constant levels during the first 6 h of elicitation.
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Results and discussion |
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Apoplast alkalinization and H2O2 accumulation
This study was initiated to identify genes that are responsive to fungal elicitor treatment of Arabidopsis cell cultures. To verify that elicitor treatment had successfully triggered a defence reaction in the cell cultures, the pH of the cell cultures was monitored and histochemical staining for H2O2 was performed following elicitor addition. There was a pH increase within 20 min of elicitor addition in the growth medium of treated cell cultures, which was not present in mock-treated cultures (Fig. 1A). Elicitor-treated cells accumulated the brown polymeric product of DAB, indicating that H2O2 had accumulated inside these cells (Fig. 1B). Inclusion of the antioxidant ascorbic acid prevented accumulation of the polymer (Fig. 1B), proving that formation of the brown compound was a specific result of production of the oxidant H2O2. Similar responses were observed using the same elicitor in maize cell suspension cultures (Chivasa et al., 2005). External medium alkalinization (Atkinson and Baker, 1989; Bolwell et al., 2002) and accumulation of H2O2 (Tenhaken et al., 1995) are well established markers for the induction of a defence response by pathogen elicitors in cell cultures. Medium alkalinization is a rapid response, thought to come about as a result of elicitor-induced inhibition of plasma membrane proton pumps (Blumwald et al., 1998). H2O2 is the most stable product of the pathogen-induced oxidative burst and serves as a local signal for activation of defence responses in the vicinity of the pathogen infection site in intact plants (Tenhaken et al., 1995; Mellersh et al., 2002; Lin et al., 2005).
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Protein analyses
2-D DIGE, a recently developed proteomic tool designed to eliminate problems arising from gel-to-gel variations in the protein profile, which limit conventional analyses with ‘single stain’ two-dimensional gel electrophoresis, was used. As a result, differences in protein abundance even below 20% can be identified with confidence, especially if multiple biological replicates are carried out to allow statistical analysis.
The 2-D DIGE employs mass and charge matched, spectrally resolvable fluorophores covalently linked to proteins via the amino group of lysine residues. To compensate for the loss of the positive charge on the lysine due to the conjugation of the dye to the lysine, the dyes are positively charged so as not to affect the isoelectric point of the protein. Three dyes (Cy2, Cy3, and Cy5) are currently available commercially and this allows up to three differentially labelled proteins to be mixed and resolved in a single two-dimensional gel. The biggest advantage of this system is that a standard sample can be included in all gels, and this is used to normalize protein abundance measurements across multiple gels in an experiment. This greatly improves inter-gel comparisons and precludes artefacts caused by differential protein loss during exit from the isoeletric focusing gel or slight variations in running conditions between gels, big drawbacks for the conventional ‘one-sample per gel’ techniques.
Six biological replicates were used and six gels, one gel for each replicate, were run. Over 1500 protein spots were detected across all gels and only a minority of these were responsive to elicitor treatment. A total of 154 protein spots changed in abundance by over 20% and 45 of these were of sufficient abundance in the gels to enable identification by mass spectrometry. A typical profile of these proteins on a 2-D gel is shown in Fig. 2 and the identified spots listed in Table 1 are annotated. Some of the proteins were present on the gel as two or more spots, suggesting the existence of alternative post-translational modifications. For example, glutathione S-transferase (At1g02930) resolved as three distinct spots (40, 41, and 42) on the gel (Fig. 2).
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Elicitor-induced transcript changes at early time-points
Protein samples for 2-D DIGE analyses were taken 24 h after adding elicitor to the cell cultures. Five proteins that had responded to elicitor treatment were selected and were investigated to see if there were any changes in their transcript abundance in the first 6 h following elicitation, since changes in transcription sometimes precede associated measurable changes in protein abundance by several hours. Using quantitative real-time reverse-transcription-PCR, it was found that transcripts for quinone reductase, glutathione S-transferase, and mitochondrial aldehyde dehydrogenase had increased higher than the control within 1 h of treatment (Fig. 3). This suggests that the increase in protein abundance for these genes was not controlled at the translational level, but at the transcription level. The vacuolar ATP synthase and thioredoxin had changed modestly at the protein level (Table 1) and it was found that there were no significant changes in transcript abundance within the first 6 h of elicitor treatment (Fig. 3). Taken together, these results show that some of the elicitor-induced changes in the proteome of Arabidopsis cell cultures are preceded by very early responses in the transcriptome. At least for three of the genes investigated, proteome changes were in agreement with transcript data.
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Molecular chaperones
Elicitor treatment down-regulated the abundance of a number of molecular chaperones that included several heat-shock proteins and three endoplasmic reticulum (ER) proteins (Table 1). Under normal growth conditions, chaperones play a role in protein folding, assembly, translocation, and degradation in diverse cellular processes (Miernyk, 1999). Their role becomes even more critical during stress responses and sometimes the ability of organisms to tolerate otherwise extreme stress conditions is solely dependent on increased expression and activity of molecular chaperones; for example, Hsc70-1 protein during heat stress in Arabidopsis (Sung and Guy, 2003). Since there was no evidence of general host protein degradation, the down-regulation of these chaperones must be of some specific significance.
However, studies conducted with whole Arabidopsis plants have revealed that ER-resident chaperones are massively increased upon pathogen attack (Schenk et al., 2000; Wang et al., 2005), prior to the synthesis of pathogenesis-related proteins required to establish an effective resistance to infection (Jelitto-Van Dooren et al., 1999). Most of these PR proteins are synthesized on the rough ER and transit through the ER en route to the vacuole or the apoplast. A build-up of ER chaperones is therefore necessary to cope with the increased demand for regulating proper folding, modification, and transport of the PR proteins in the secretory system. A failure to increase molecular chaperones is believed to cause a build-up of unfolded and misfolded proteins, leading to cell death as demonstrated by the hypersensitivity to salicylic acid analogues of gene knockout mutants of the ER chaperone BiP2 (Wang et al., 2005). In this vein, the down-regulation of the ER chaperones BiP1 and BiP2 in the treated cell cultures observed could possibly be a part of the mechanism employed to allow the inauguration of programmed cell death (Lam et al., 2001), an integral component of the defence response. The reason why an increase in most of these chaperones was not seen could be that all the cells in a cell suspension culture experimental system come into contact with the elicitor and are thus equivalent to directly invaded cells in whole plants that usually undergo a hypersensitive cell death.
Thus, the present results confirm previous findings showing the important role of the protein secretory system in plant pathogen defence. Down-regulation of ER and non-ER chaperones observed in the present study suggests that the whole cellular chaperone system could be the target for activation of the hypersensitive cell death in cells that have come into direct contact with pathogen elicitor molecules. This is supported by reports that down-regulation of Arabidopsis heat-shock cognate Hsc70-1 (Sung and Guy, 2003), also identified in this study, and tobacco lumenal binding protein (Leborgne-Castel et al., 1999) by antisense technology is lethal even under normal growth conditions.
Mitochondrial proteins
It was noted that several spots identified as mitochondrial proteins were up-regulated by elicitor treatment except for the putative isocitrate dehydrogenase, which was down-regulated (Table 1). Pathogen elicitors affect mitochondrial functions (Xie and Chen, 2000) and the elicitor-induced programmed cell death signals are, in part, generated by this organelle (Lam et al., 2001). Pathogen-induced accumulation of reactive oxygen species is pivotal to the induction of a defence response and the mitochondrion was recently identified as one of the organelles that generates massive bursts of reactive oxygen during elicitor treatment (Krause and Durner, 2004). The present results reveal that the effects of pathogen elicitor treatment on plant mitochondrial metabolism are not only restricted to cytochrome c (Krause and Durner, 2004) and the alternative oxidase (Chivasa and Carr, 1998), but extend to other mitochondrial proteins reported here. Perhaps the loss of cytochrome c and engagement of the alternative oxidase, which disrupts ATP synthesis, cause the cells to increase all the other mitochondrial enzymes in an attempt to prevent or limit the inevitable cell death caused by ATP depletion.
Antioxidative proteins
Eleven elicitor-responsive protein spots were identified as enzymes that are integral components of the machinery that protects cells from oxidative damage. These included several glutathione S-transferases, two peroxidases, a putative thioredoxin, and two reductase enzymes (Table 1). It was noted that the most robust response was within this group of proteins, with the highest response being of a glutathione S-transferase that was induced by over 30-fold. Although elicitor treatment generates a rapid oxidative burst, which is required for activation and establishment of signalling cascades of the defence response, the host plant has to curtail the propagation of toxic products in order to localize cell death in the fashion characteristic of the hypersensitive response. Obviously the host deploys a number of antioxidant and detoxifying enzymes to achieve this and the present results reveal the diverse enzymes mobilized by elicitor-treated Arabidopsis cells.
The protective effects of the Arabidopsis NADP-dependent oxidoreductase (P1) against oxidative stress were demonstrated in yeast and this gene is also induced in Arabidopsis by treatment with oxidative stress-inducing compounds such as paraquat, menadione, and diamide (Babiychuk et al., 1995). Detoxification of harmful lipid peroxides, products of the action of reactive oxygen species on membrane lipids, is performed by glutathione S-transferases and phospholipid hydroperoxide glutathione peroxidase. Previous studies have shown that transcripts of the glutathione S-transferase genes, At1g02930 and At4g02520, accumulate in plants challenged with Peronospora parasitica or treated with salicylic acid (Wagner et al., 2002). The Arabidopsis phospholipid hydroperoxide glutathione peroxidase is also up-regulated by exposure to other treatments generating oxidative stress, such as NaCl and Al3+ (Sugimoto and Sakamoto, 1997).
The cytosolic L-ascorbate peroxidase 1 (At5g16970) and a putative thioredoxin (At4g04950) were down-regulated by elicitor treatment (Table 1). Heterologous expression of Arabidopsis thioredoxins in yeast oxidant-hypersensitive mutants (Mouaheb et al., 1998; Issakidis-Bourguet et al., 2001) demonstrated their role in defence against oxidative damage. The role of ascorbate peroxidase in defence against oxidative stress is well established (Davletova et al., 2005). Differential expression of the putative thioredoxin ascorbate peroxidase proteins reveals that oxidant protection conferred by these proteins is regulated in Arabidopsis during elicitor treatment.
Ethylene and jasmonate pathways
Two genes important in the biosynthesis of vital endogenous signalling molecules, jasmonic acid and ethylene, were induced by elicitor treatment. One of the intermediate metabolites in the synthetic pathway of jasmonic acid is 12-oxophytodienoic acid, which is reduced to 3-2(2'(Z)-pentenyl) cyclopentane-1-octanoic acid by 12-oxophytodienoate reductase, identified in this study (Table 1). The ethylene-forming enzyme (ACC oxidase) catalyses the terminal reaction in ethylene biosynthesis; the oxidation of 1-aminocyclopropane-1-carboxylic acid to ethylene. The increase in accumulation of these proteins suggests that elicitor treatment of Arabidopsis cells induces the biosynthesis of jasmonic acid and ethylene. Both hormones are crucial defence signalling molecules required for activation of certain defence proteins (Penninckx et al., 1998).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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In addition to the protein classes discussed above, several other proteins ranging from proton pumps and enzymes of amino acid metabolism, glycolysis, and protein translation were also differentially expressed during pathogen elicitor treatment (Table 1). The diversity of the biochemical pathways responding to elicitor treatment may reflect a switch from primary metabolism to primed defence secondary metabolism. Non-essential pathways probably need to be down-regulated so that metabolites are funnelled into biochemical pathways leading to the production of defence proteins and compounds. Switching off of less important pathways during stress could be achieved by direct down-regulation of the pathway components or indirectly by repressing the corresponding molecular chaperones as was seen here. Similarly, essential pathways can be up-regulated directly by increasing the protein components and up-regulating the cognate chaperones. However, a full picture of the elicitor-induced response will require subcellular proteomic analyses so that changes in protein abundance can be related to the respective organelles. This will clarify the role of particular protein isoforms and organelles, as has been attempted here for mitochondrial proteins. Additionally, time-course experiments need to be performed so that individual proteins or pathways can be investigated over the duration of the defence response, from initiation to termination.
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Acknowledgements |
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We wish to thank the BBSRC and the ICBL for grants that enabled setting up the Durham Proteomic facility. We are grateful to the Royal Society for a fellowship to support BKN.
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Abbreviations |
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DAB, 3,3'-diaminobenzidine; 2-D DIGE, two-dimensional difference gel electrophoresis; ER, endoplasmic reticulum; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PMT, photon multiplier tube; PR, pathogenesis-related.
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References |
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