JXB Advance Access originally published online on July 25, 2006
Journal of Experimental Botany 2006 57(11):2847-2865; doi:10.1093/jxb/erl043
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Transcriptional analysis of calcium-dependent and calcium-independent signalling pathways induced by oligogalacturonides
1Department of Biology, University of Padua, Via Ugo Bassi 58b, I-35131 Padova, Italy
2Department of Biology (Area 9), University of York, PO Box 373, York YO10 5YW, UK
*To whom correspondence should be addressed. E-mail: fjm3{at}york.ac.uk
Received 5 March 2006; Accepted 9 May 2006
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Abstract |
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-1,4-linked oligogalacturonides (OGs) are pectic fragments of plant cell walls that are able to induce defence and developmental responses. To understand plant responses to OGs at the transcriptional level, changes in gene expression were examined using oligonucleotide-based microarrays that cover almost the entire Arabidopsis transcriptome. In suspension-cultured Arabidopsis thaliana (L.) Columbia hypocotyl cells, approximately 4% of the total transcriptome exhibited significant change in abundance in response to treatment with OGs for 2 h. Steady-state changes in the abundance of transcripts encoding stress- and disease-related proteins, signalling components, and transcription factors were particularly noteworthy. As in other plant cell types, OGs elicit a rapid, but transient, elevation in cytosolic free Ca2+. The Ca2+ transient can be abolished by the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and by the Ca2+ channel inhibitor La3+, thereby facilitating a distinction between Ca2+-dependent and -independent transcriptional responses. Among the 244 transcripts that were up-regulated by OGs, the response of 93 (38%) was selectively sensitive to abolition of the Ca2+ transient. These OG-up-regulated, Ca2+-dependent transcripts included two noteworthy classes, the first comprising genes involved in cell wall modification following pathogen attack, and the second consisting of genes involved in the biosynthesis of jasmonate and C6 volatile compounds. These results support the notion of an important role for cytosolic Ca2+ signalling in jasmonate biosynthesis following OG perception. Promoter analysis of OG-induced, inhibitor-sensitive and -insensitive genes identified several putative cis-elements that might be involved specifically in Ca2+-dependent transcriptional regulation. Key words: Arabidopsis, calcium, oligogalacturonides, signalling, transcriptomics
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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During plant–pathogen interactions oligosaccharide fragments, generated by the depolymerization of the parietal polysaccharides (Côté and Hahn, 1994; John et al., 1997), can elicit defence responses such as cell wall fortification through oxidative cross-linking of cell wall polymers, the generation and accumulation of reactive oxygen species (ROS) (Lamb and Dixon, 1997; Cessna and Low, 2001), the production of antimicrobial secondary metabolites such as phytoalexins, and the synthesis of pathogen-related proteins. Among the pectic breakdown fragments,
-1,4-linked oligogalacturonides (OGs) have been shown to be especially potent defence response elicitors (Côté and Hahn, 1994; Ridley et al., 2001). At concentrations lower than those pertaining in defence response signalling, OGs also have profound effects on plant development through interference with auxin-induced cell elongation, flower development, and root organogenesis (Côté and Hahn, 1994; Bellincampi et al., 1996). In addition, OGs have been shown to stimulate stomatal and pericycle cell differentiation (Altamura et al., 1998). Apart from pathogen attack, the action of herbivores may also lead to the production of OGs, either through mechanical tissue damage or through OG release from cell wall pectin by the introduction of polygalacturonase-containing saliva into the wounding site (Miles, 1999). Not all OGs are equally capable of generating cellular responses. Oligomers with a degree of polymerization (DP) between 10 and 15 have been shown to be the most potent inducers of defence responses (Darvill et al., 1992; Van Cutsem and Messiaen, 1994), a property that has generally been attributed to their ability to form hetero-oligomeric complexes with Ca2+ (Liners et al., 1992). Nevertheless, smaller oligomers have also been shown to generate defence responses in plants, for example, in potato where OGs with a DP of 2–4 induce resistance against Erwinia carotorova (Wegener et al., 1996). OGs with a DP less than 8 can trigger plant cell death during tissue decay induced by E. carotovora in potato (Weber et al., 1996), ethylene production (Simpson et al., 1998), induction of genes involved in metabolism and/or synthesis of jasmonic acid (Norman et al., 1999), and the accumulation of protease inhibitors (Moloshok et al., 1992).
Currently, very little is understood regarding the intermediate processes that couple the perception of OGs to cellular responses such as the induction of defence mechanisms against pathogens. Coupling of primary stimuli, such as OGs, to cellular targets often involves intracellular messengers of which Ca2+ is considered one of the most versatile. The capacity of Ca2+ to couple a wide range of extracellular signals to meaningful responses relies on generating Ca2+ transients with unique stimulus-specific kinetics (calcium signatures: Sanders et al., 2002). Previous work from this laboratory (Navazio et al., 2002) and from others (Van Cutsem and Messiaen, 1994; Chandra et al., 1997) has shown that Ca2+ signalling is involved in OG-induced signal transduction. In soybean cells exposure to OGs invoked a rapid and transient increase of cytosolic Ca2+ that appeared to precede both alkalinization of the extracellular medium (Felix et al., 1993) and H2O2 production (Chandra et al., 1997; Cessna and Low, 2001; Navazio et al., 2002). Both extra- and intracellular stores are likely to contribute to the Ca2+ signal. Pretreatment of cells with the Ca2+ channel blocker La3+ or the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) completely abolished the Ca2+ transient induced by OGs. The effect of TBB suggests an upstream phosphorylation event is essential for the generation of the Ca2+ signal. Exposure to TBB also abolished the emergence of extracellular H2O2.
Although our knowledge of the initial stages of OG-induced signalling is fragmentary, the pathways by which early events of the signal cascade lead to meaningful cellular responses is even less clear. A productive strategy to identify downstream targets of OG-based stimuli is to query the transcriptome for changes after exposure to OGs. Some studies into the regulation of transcripts in response to pathogens and wounding have been reported (Cheong et al., 2002). However, none of these has specifically focused on the role of OGs in such processes, nor were such studies carried out genome-wide. Thus, a microarray approach was used, offering comprehensive coverage of the Arabidopsis transcriptome to identify transcripts that are rapidly modulated after exposure of mesophyll suspension cultures to OGs. Moreover, selective abolition of the OG-induced Ca2+ transient by TBB and La3+ made it possible to distinguish between Ca2+-dependent and Ca2+-independent pathways that exert control over gene transcription downstream of the OG stimulus.
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Materials and methods |
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Plant material
Arabidopsis thaliana (L.) Columbia hypocotyl-derived cell-suspension cultures were maintained at 24 °C on a rotary shaker at 80 rpm under an 18 h photoperiod at 80–100 µmol m–2 s–1 light intensity. Cells were subcultured every week with a 10% (v/v) inoculum in Murashige and Skoog (MS) liquid medium supplemented with 3% (w/v) sucrose, 0.5 µg ml–1 2,4-dichlorophenoxyacetic acid, and 0.25 µg ml–1 6-benzylaminopurine.
Production and isolation of oligogalacturonic acids
OGs were obtained according to the method described by Simpson et al. (1998). Briefly, 5 g of polygalacturonic acid (PGA) from orange pectin (Sigma), dissolved in 0.5% ammonium oxalate (5 mg ml–1), were repeatedly dialysed against dH2O and concentrated by vacuum evaporation, then de-esterified with cold alkali and freeze-dried. De-esterified PGA was dissolved in dH2O (5 mg ml–1) and heated to 37 °C, after which 0.03 mU mg–1 of Aspergillus niger polygalacturonase (Sigma) was added. After incubation for 1 h, digestion was stopped by heating to 100 °C. To isolate oligomers with a DP of 5–15 the digested PGA was selectively precipitated with ethanol and sodium acetate (Spiro et al., 1993). The precipitate was redissolved in dH2O and separated by anion exchange chromatography on a QAE-Sephadex A-25 matrix (Pharmacia, 2.5 cmx60 cm) equilibrated with 50 mM ammonium formate pH 9. OGs were eluted using a linear gradient running from 250 mM to 1000 mM ammonium formate pH 9 at a flow rate of 2 ml min–1 (total volume 4.0 l). Fractions (8 ml) were assayed for their uronic acid content by the m-hydroxydiphenyl method (Van den Hoogen, 1998) using galacturonic acid as a standard. Individual peaks were pooled, diluted 1:1 with dH2O, and freeze-dried several times to remove the ammonium formate. The size and purity of the OG oligomers eluted in each peak was determined by MALDI mass spectrometry: samples (125 pmol) were mixed on the target plate with 2,5-dihydroxybenzoic acid and allowed to dry. The target spots were then recrystallized in 0.5 ml ethanol (Harvey, 1993). Positive ion MALDI mass spectra were recorded with a PerSeptive Biosystems Voyager Elite time-of-flight mass spectrometer (nitrogen laser, 337 nm) operating in the reflectron mode. The delayed-extraction ion source was operated with a 75 ns delay, the extraction voltage was 20 kV and the grid voltage was set at 65%.
Finally, individual peaks in the range of DP 10–15 were pooled and de-salted on a 500 ml column of Sephadex G-25 matrix (Pharmacia), equilibrated and eluted with dH2O.
Reconstitution of aequorin
Aequorin reconstitution was done as previously described (Navazio et al., 2002) for soybean cells and entailed incubation of 10-d-old transgenic Arabidopsis cells with 5 µM coelenterazine added to the cell culture medium, overnight in darkness. Cells were then washed three times with 10 vols of fresh hormone-free culture medium and used after 30 min.
Aequorin luminescence measurement and Ca2+ calibration
Suspension-cultured cells were transferred to a purpose-built chamber placed in close proximity to a low-noise photomultiplier, with a built-in amplifier discriminator (Navazio et al., 2002). All measurements were performed at room temperature in a final volume of 50 µl containing approximately 3 mg (fresh weight) of reconstituted cell-suspension culture. Treatment with OGs was carried out by injecting an equal volume of 2-fold-concentrated stock solutions (dissolved in the basal cell-culture medium) through the luminometer port into the cell-suspension culture, using a light-tight syringe. All experiments were terminated by discharging the remaining aequorin pool with 0.33 M CaCl2 in 10% (v/v) ethanol. The output of the discriminator was captured by a Thorn-EMI photoncounting board and stored in an IBM-compatible computer for further analyses. The aequorin luminescence data were calibrated off-line into [Ca2+] values, using a computer algorithm based on the Ca2+ response curve of aequorin, as described by Brini et al. (1995).
Microarray hybridization and analysis
10-d-old Arabidopsis cells were treated with either (i) oligogalacturonides (200 µg ml–1, 2 h), (ii) oligogalacturonides plus TBB (50 µM, 10 min prior to addtion of OGs), (iii) TBB only, or (iv) oligogalacturonides plus La3+ (3 mM, 10 min prior to addtion of OGs), and total RNA was extracted using RNeasy columns (Qiagen, UK) from treated and control cells. Total RNA of three independent growth cultures was pooled for each treatment and this procedure was repeated three times, i.e. a total of 12 (three for each treatment) microarrays was hybridized. For each hybridization, approximately 100 µg of total RNA was primed with Random 15-primer (0.5 µg µl–1; Operon) and reverse-transcribed with Superscript II (Invitrogen). Fluorescent labelling was achieved by replacing dCTP in the dNTP mix (Sigma) with Cy3-dCTP and Cy5-dCTP (Amersham, UK). Labelled cDNA was cleaned on a QIAquick spin column (Qiagen, UK). Arabidopsis Oligonucleotide Microarrays (http//ag.arizona.edu/microarray), using the Arabidopsis Qiagen-Operon Genome Oligo Set that represents around 26 000 coding sequences, were used for hybridization. Array cross-linking, hybridization, and post-hybridization washes were carried out as described by the manufacturer (http//ag.arizona.edu/microarray).
Arrays were scanned using an Axon (Axon Instruments, Braintree, UK) scanner and initial array analysis was carried out with ScanAlyze2 software (http://rana.lbl.gov/EisenSoftware.htm). Background subtraction, global normalization of fluorescence signals and lowess signal correction were performed using SNOMAD software available at http://pevsnerlab.kennedykrieger.org/snomadinput.html. Signals were designated as ‘present’ when a signal background ratio of >1.5 was found in at least one channel. Global mean normalization was carried out across microarray surfaces and local mean normalization across element signal intensity. After normalization and log2-transformation, signal averages and the standard deviations for signal ratios of the three replica experiments were calculated. Transcripts were included for analysis and annotated as significantly regulated when the following criteria were met: (i) a ‘present’ signal on all three replicas, (ii) a signal ratio average of four (log22) or more between treated and control transcripts, and (iii) a ratio between the ratio average and the standard deviation greater than 1+0.5xstandard deviation. The fold-change cut off criterion (four) was based on the distribution of fold-changes observed in control data such that the number of treatment-induced false positives is 5% or lower.
RT-PCR analysis
A proportion of the RNA obtained for microarray studies was used for RT-PCR. After DNase I treatment (Ambion Ltd., UK), 5 µg of total RNA was primed with Random Decamers (Ambion), reverse-transcribed with PowerScript Reverse Transcriptase (Clontech, USA) and diluted 1:5. Relative-quantitative RT-PCR was performed with 5 µl diluted first-strand cDNA, using 18S rRNA as an internal standard (QuantumRNA Universal 18S Internal Standards Kit, Ambion Ltd., UK). The 18S Primers:Competimers ratio was established as 1:9. The primers used to obtain gene amplicons (
200 bp) were: AOS (At5g42650) 5'-ACGCTCCGGGTTTGATCACTAAATG-3', 5'-CCCAATTTATCGGCTTCAACGAGAA-3'; LOX2 (At1g72520) 5'-GAGTCGTGCTTCACTGCTGGTCAAT-3', 5'-ATAAGAGACCGTCGTTGGCGTATGG-3'; ACS (At4g11280) 5'-TGGTGGCT-TTTGCAACAGAGAAGAA-3', 5'-ACGCATCAAATCTCCACAAAGCTGA-3'; ACO (At1g06650) 5'-AGTTCCACGCATCTTTCATCATCCA-3', 5'-TGATCACCTGGAAGAAACCCCACTT-3'; MAPK3 (At3g45640) 5'-ATGCGAAAAGATACATCCGGCAACT-3', 5'-TCATCATTCGGGTCGTGCAATTTAG-3'; Disease Resistance (At5g41750) 5'-TCGGTAGGTAAGGGGGCTTTTGAAG-3', 5'-AATTTTGACGAGATGTTCCGGGTTG-3'; MAPKKK5 (At5g66850) 5'-CTGATTTCGGCATGGCTAAACACCT-3', 5'-CAAGGAGGCTTCCCAGTGAACATCT-3'; Multi drug resistance (At4g25960) 5'-AAGGCTGGTGAGATTGCAGAAGAGG-3', 5'-ACGAGCAAGGCCCAAGATAGAAACA-3'. The thermocycler was programmed with the following parameters: 20 s at 94 °C, 30 s at 68 °C, and Advantage 2 Polymerase Mix (Clontech) was used as Taq Polymerase. Densitometric analysis of ethidium bromide-stained agarose gels (0.5 µg ml–1) was performed using Quantity One software (Bio-Rad).
Promoter cis-element analysis
For the detection of putative regulatory cis elements in the promoter regions of coregulated transcripts, 5' upstream sequences of up to 800 bp (avoiding overlap with preceding coding sequences) were uploaded at the ‘Regulatory Sequences Analysis Tools’ service at ‘http://rsat.ulb.ac.be/rsat/’. Sequences were queried using algorithms (van Helden et al., 1998) to detect over-represented strings of 4–8 nucleotides searching both DNA strands. The P-value represents the probability for the number of detected motifs to occur relative to the expected number of occurrences based on the motif distribution in the background dataset which contains all Arabidopsis 5' upstream sequences. A significance cut-off of P <10–5 was used in all analyses. Identified putative promoter elements were used to query Arabidopsis cis-element databases such as PlantCARE (http://oberon.fvms.ugent.be:8080/PlantCARE/), Agris (http://arabidopsis.med.ohio-state.edu/AtcisDB/), and Atprobe (http://exon.cshl.org/cgi-bin/atprobe/atprobe.pl) for known functions.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Arabidopsis cells show a large OG-induced Ca2+ transient
It has previously been shown that a cytosolic Ca2+ transient is rapidly generated in soybean cells after exposure to OGs (Navazio et al., 2002). Figure 1 shows that in Arabidopsis mesophyll suspension culture cells a similar Ca2+ signal occurs after the addition of 10 µg ml–1 OGs (DP 10–15) to the medium. Furthermore, as was observed for soybean cells, pretreatment with 50 µM TBB completely abolished the Ca2+ transient. This observation suggests that, in Arabidopsis, too, protein kinase-dependent phosphorylation might be involved in the early stages of OG signalling.
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Functional dissection of transcriptional responses to OGs
To study gene expression in response to treatment with OGs, oligonucleotide-based microarrays were used that fully cover the Arabidopsis transcriptome. The arrays were probed with cDNAs derived from four different treatments to enable comparison on individual microarrays between (i) ‘control’ and ‘OG-treated’ cells, (ii) ‘control’ and ‘OG plus TBB-treated’ cells, (iii) ‘control’ and ‘TBB-treated’ cells, and (iv) ‘control’ and ‘OG plus La3+-treated, cells. The first condition (i) allows determination of how the presence of OGs impacts on the entire transcriptome. In addition, it is possible to distinguish within the results those changes in transcript level that are due to Ca2+-dependent and Ca2+-independent pathways by comparing the outcome of condition (i) with conditions where the initial Ca2+ transient is inhibited. TBB has been shown previously to remove the Ca2+ transient totally and thus condition (ii) should provide insight into OG-induced Ca2+-dependent and Ca2+-independent processes. Non-specific effects of the protein kinase inhibitor were accounted for and subsequently eliminated from analysis by including condition (iii). However, it can not be ruled out that TBB may have specific effects during OG-induced transcriptional regulation that do not involve the inhibition of the early Ca2+ signal. A second treatment (condition iv), which has also been shown to eliminate the early Ca2+ signal (Navazio et al., 2002), was therefore included. Only those transcripts that showed sensitivity to both condition (ii) and (iv) were considered for further analysis. For each treatment, RNA was isolated after 2 h and all data represent three independent experiments for each treatment.
Figure 2 shows a Venn diagram representing the total number of transcripts, 1237, that was changed by the treatments. For clarity, conditions (ii) and (iv), which both act to eliminate the Ca2+ transient and largely overlapped, are represented as one ‘OG plus inhibitor’ dataset. To ensure that analysis was restricted only to significantly changed transcripts, a robust threshold criterion of 4-fold (i.e. 2 on a log2 base) was applied to qualify for description as a change in transcript abundance. Overlapping regions only contain transcripts that were significantly affected in the same direction (i.e. up or down). Of the 1237 transcripts that exceeded this threshold, 320 transcripts (26%) responded specifically to ‘OG’ treatment, 424 (34%) were responsive to both ‘OG’ and ‘OG plus inhibitor’ treatments, whereas 330 transcripts responded solely to the ‘OG plus inhibitor’ treatment. Condition (iii), ‘TBB’ treatment, showed that 158 transcripts responded to TBB in the absence of OGs of which 38 overlapped with the ‘OG plus inhibitor’ data. These false positives were removed from the ‘OG plus inhibitor’ dataset in all subsequent analyses. Conditions (ii) and (iv) were compared to identify transcripts that responded to OGs (condition i) but not to ‘OG plus TBB’ (condition ii) and ‘OG plus La3+’ (condition iv) and only transcripts that were sensitive to both TBB and La3+ were annotated as inhibitor-sensitive and included in the subsequent analyses. Accession numbers and numerical data for all transcripts can be found in the supplementary data files at JXB online.
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Independent confirmation of array data using RT-PCR (Table 1) for a number of key transcripts (see below) shows an overall agreement between the two methods although transcript changes were generally found to be less pronounced when assessed by RT-PCR.
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The OG-responsive, inhibitor-sensitive fraction and the OG-responsive, inhibitor-insensitive fraction totalled 746 transcripts. Of these, a total of 413 could be classified into specific functional categories. The predominant categories are listed in Table 2 and represent transcripts encoding proteins involved in signal transduction (e.g. protein kinases, phosphatases, calcium binding proteins, G-proteins, ethylene and jasmonate signalling pathways), gene transcription (e.g. ethylene binding factors, bHLH, bZIP, MADS, Myb, Myc, NAC/NAM, Zinc finger, Heat shock, WRKY), stress and disease (e.g. cytochrome P450, disease resistance proteins, chitinases), and cell wall modification (e.g. glycosyl hydrolases, polygalacturonases).
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It is noteworthy that treatment with OGs increased the transcript levels of many typical pathogen-induced genes, including plant disease resistance genes (R genes) involved in the detection of pathogens. Many of the up-regulated R genes encode proteins containing nucleotide-binding sites (NBS), toll/interleukin receptor (TIR) domains, and leucine-rich repeat (LRR) domains. Among the up-regulated TIR-NBS and TIR-NBS-LRR type disease resistance transcripts are RPP1-WsA-C and RPP1-WsB-like gene products, but also many that have not been characterized.
Post-translational regulation through phosphorylation during the OG response appears to be prevalent judging by the many phosphatase and kinase encoding transcripts that are affected by OG treatment. These included mitogen-activated protein (MAP) kinases, plant receptor kinases (PRKs), or receptor-like kinases (RLKs), and serine/threonine kinases. MAP kinase cascades have been shown to be involved in pathogen responses, for example, in tobacco cells after OG treatment (Lebrun-Garcia et al., 1998), whereas various transcription factors can also be MAPK targets (Tena et al., 2001). The present data show particularly that MAPK3 and a putative MAPK, which is very similar to MAPK4 that was previously reported to be induced by wounding (Cheong et al., 2002), were significantly up-regulated after OG treatment. Activation of MAPK3 by fungal elicitors has been observed in alfalfa cells (Cardinale et al., 2000). Further up-regulated MAPKs included MAPKK9, MAPKKK19, and MAPKKK8 (MEKK1). Down-regulated MAPKs included MAPK8, a MAPKKK5, and a MAPKK1.
The transcript level of one protein phosphatase 2C (PP2C) isoform was greatly increased by OG treatment. PP2C is considered to regulate various signalling pathways (Rodriguez, 1998) and is a specific MAPK inactivator (Tena et al., 2001). Up-regulation of PP2C could therefore lead to post-translational reduction in activity of selective MAPK cascades, in addition to possible effects of reduced transcript levels of MAPK-type kinases such as MAPK8, MEK, and MAPKKK5.
Exposure to OGs has been demonstrated to activate jasmonate (Doares et al., 1995) and ethylene (Simpson et al., 1998) signalling pathways and the interaction between these hormones determines the type of response to pathogen attack or wounding, including the expression of particular defence proteins such as PR1b, PR5 (osmotin) and PDF2.1 (Table 2; Xu et al., 1994; Penninckx et al., 1998). Transcripts involved in the biosynthesis of both hormones were also increased after exposure to OGs. These included genes encoding aminocyclopropane 1-carboxylic acid synthase (ACS-6) and aminocyclopropane 1-carboxylic acid oxidase (ACC oxidase), two enzymes required for ethylene biosynthesis, and for genes encoding lipoxygenase (LOX) and allene oxide synthase (AOS), two enzymes required for jasmonate biosynthesis.
Functional categories of genes show differential inhibitor sensitivity
For many transcripts that were significantly regulated by OG treatment, this regulation was prevented by the inclusion of a Ca2+ signal abolishing inhibitor. Although our discrimination criteria were based on one specific cut-off value, Table 2 shows that in most cases there is a large difference between the observed ratio value in the ‘OG’ condition and the ‘OG plus inhibitor’ condition, giving extra confidence to our classification of inhibitor-sensitive and inhibitor-insensitive transcripts.
The relative proportions of inhibitor-sensitive and -insensitive transcripts varied greatly across functional categories of OG-responsive genes, (Table 2). To identify which functional categories are likely to require an early Ca2+ signalling event for transcriptional regulation, data were therefore analysed for all 413 functionally annotated genes with respect to inhibitor sensitivity. In the complete dataset of 244 up-regulated genes, 97 were found to be inhibitor-sensitive and 147 are inhibitor-insensitive. Among down-regulated genes, 65 were inhibitor-sensitive out of a total of 169, whereas 104 genes were insensitive.
Data from each functional category were analysed to test whether the binomial distribution (see http://fonsg3.let.uva.nl/Service/Statistics/Binomial_proportions.html) of inhibitor-sensitive and -insensitive genes in that particular category deviated significantly from the distribution in the reference dataset consisting of 1117 transcripts of which 320 were inhibitor-sensitive. Table 3 reports those categories that showed a significant change from the null hypothesis P1=P2 (i.e. binomial distribution of category is not significantly different from the binomial distribution of the background). Among categories of genes that were up-regulated by OGs, those involved in cell wall modification were significantly inhibitor-sensitive (Table 3; 9 out of 17 genes). It is noteworthy that this was the case only for the group of cell wall modification genes that was transcriptionally up-regulated by OGs and did not apply to down-regulated genes. Conversely, Nac/Nam type transcription factors were found to be significantly inhibitor-sensitive, but only for the subset that was down-regulated in response to OGs. Of particular interest is the finding that the jasmonate synthesis pathway is disproportionately inhibitor-sensitive, with three out of four OG-up-regulated jasmonate production transcripts showing inhibitor sensitivity. However, the small number of transcripts in this group only yielded a significant P value (0.041) at the 5% level. In all other categories, no evidence for significant deviation from the background distribution of inhibitor sensitivity was obtained, including disproportionate inhibitor insensitivity. Thus, our analysis suggests that two of the processes that contribute to rapid OG responses, modification of the cell wall and the induction of jasmonate synthesis, may require an upstream Ca2+ signal. By contrast, ethylene synthesis, the oxidative burst, phosphorylation, and the transcriptional regulation of stress-related genes and induction of many transcription factors themselves rely on both Ca2+-dependent and Ca2+-independent upstream events.
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To analyse further which OG-induced signalling components require upstream Ca2+ transients, the data were re-ordered (Table 4) into three major classes of ‘wounding’, ‘jasmonate’, and ‘ethylene’-related genes, since several reports have described a close causal link between wounding, the production of OGs and the induction of jasmonate and ethylene signalling pathways (Schenk et al., 2000; Cheong et al., 2002; Van Zhong et al., 2003; DRASTIC Data Base: http://www.drastic.org.uk/). For example, the dataset of jasmonate signalling-related genes contains, in addition to those directly involved in jasmonic acid synthesis, genes encoding kinases, glycosyl hydrolases, and transcription factors that are known to play a role in jasmonate signalling or known to be transcriptionally regulated by jasmonate treatment. Among transcripts that were significantly regulated by OGs, 101 genes were recognized as associated with wounding, 56 with jasmonate signalling, and 46 with ethylene signalling. To test whether Ca2+ dependence was restricted to jasmonate synthesis per se or whether it pertains to a broader range of targets in either the jasmonate-associated gene group or the other groups, the same binomial analysis as described above was applied. For all wounding-associated genes the distribution of inhibitor-sensitive and -insensitive genes did not deviate significantly from the distribution found in the background set. A similar finding was made regarding ethylene synthesis and ethylene signalling-related genes. However, both categories of up-regulated and down-regulated jasmonate-associated genes exhibited a highly significant inhibitor sensitivity (Table 4) with an overall significance score for all jasmonate-associated genes of P
1.2e–5.
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These results suggest that, in addition to the gene products responsible for jasmonic acid metabolism (Tables 2, 3) the jasmonate signalling network itself is Ca2+-dependent. By contrast, the wounding response and both ethylene synthesis and the induction of many ethylene signalling associated genes appear to require both Ca2+-dependent and Ca2+-independent components.
To confirm our findings, an alternative analysis was carried out on the OG responsive transcripts that is not based on fold-change criteria but calculates a ‘Rank Product’ for each transcript (Breitling et al., 2004). Data in the supplementary file ‘RankProduct_SupplData.txt’ at JXB online not only show a high ranking for the relevant transcripts involved in ethylene and jasmonic acid biosynthesis but also a very low probability of being false positives.
Identification of putative promoter cis-elements
The rapid changes in the level of many transcripts that follows exposure to OGs implies that transcriptional regulation of some or many of these genes might rely on common regulatory motifs in their promoters. To determine whether such common motifs or cis-elements are present, the 5' upstream regions of OG-responsive genes were queried for overrepresented motifs. Analyses were carried out for total data sets (i.e. all up-regulated or down-regulated genes), respective functional categories, and across inhibitor-sensitive and -insensitive subcategories. Our particular interest was to identify putative motifs that were associated with either Ca2+-dependent or Ca2+-independent categories.
Table 5 lists for the various data sets putative motifs with a P score <10–5. Most of the identified motifs are previously unrecognized and their significance has yet to be established. A few putative motifs, for example, 5'-ACCACCGT-3', 5'-AGTTTTAT-3', and 5'-GGATAACA-3', occur only in inhibitor-sensitive categories and may therefore involve Ca2+ signalling for the activation of their associated transcription factors.
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Of the several motifs that were found and were previously described, the ‘Dof core’ motif from maize was the most prevalent. Dof proteins are plant transcription factors that contain conserved single Zn-finger motifs. A large number of Zn-finger type transcription factors was found to be up-regulated by OGs (Table 2) and Dof proteins have previously been shown to be elicitor responsive, for example, ERDP from Pisum sativum (Lijavetzky et al., 2003). Further known elements included: ‘Pollen lelat’, one of two co-dependent regulatory elements responsible for pollen-specific activation of the tomato lat52 gene involved in pollen development; ‘CAAT-boxes’, common promoter elements that act as transcription enhancers; an ‘I-box’ that is believed to play a role in light response; and ‘ABRElaterd1’, an ABA-responsive element-like sequence required for expression of erd1 (early responsive to dehydration).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Formation of cell wall degradation products in the form of OGs provides a potent cue for plant cells to respond to attack by pathogenic micro-organisms or herbivores. Processing of this response includes OG perception, subsequent signal transduction, and the activation of cellular targets by transcriptional regulation. Very little is known regarding the details of the signal transduction events between OG perception and downstream targets, but several reports have shown the involvement of Ca2+ as a potential second messenger (Messiaen and Van Cutsem, 1994; Chandra et al., 1997; Navazio et al., 2002). Yet the significance of the intermediate Ca2+ signal and its downstream targets remain largely unknown.
In a previous study (Navazio et al., 2002), it was shown that, in soybean cells, the OG-induced Ca2+ transient can be completely abolished by the administration of the protein kinase inhibitor TBB, suggesting that a phosphorylation event is essential for the generation of the Ca2+ signal. The current work demonstrates that in Arabidopsis as well, the Ca2+ transient induced by OGs is completely abolished by TBB pretreatment (Fig. 1). Therefore, using a microarray-based approach with TBB as a diagnostic tool, it was possible to dissect OG-induced signalling into Ca2+-dependent and Ca2+-independent components and to establish the role of OGs in the transcriptional regulation of targets involved in the pathogen response.
Using our significance criteria (see Materials and methods) around 1080 transcripts, or 4% of the Arabidopsis transcriptome, were found to change in abundance within 2 h exposure to OGs. Apart from a large number of unknown and hypothetical proteins, this group of genes predominantly encoded stress- and disease-related proteins, signalling components, and transcription factors. Within these groups many transcript levels changed substantially, even after the relatively short period of OG exposure. Transcripts that changed most markedly in abundance included those related to disease resistance proteins, kinases, and cytochrome P450.
Analysed on the basis of sensitivity to inhibitors that abolish the Ca2+ transient, specific functional categories were found that appear to be more Ca2+ dependent. Although these data do not provide direct evidence for altered protein activity, they may form an indication that specific biochemical functions do require an initial Ca2+ signal. First, many of the genes involved in post-pathogen attack cell-wall modification, for example, lignin formation, fail to be induced after inhibitor treatment and therefore seem to require an upstream Ca2+ signal. The group of genes involved in the biosynthesis of jasmonate (AOS and LOX) and of C6 volatile compounds showed sensitivity in three out of four. The latter group is known to be involved in many signal pathways, for example, those occurring after wounding (Leon et al., 2001). By contrast, transcriptional regulation of very few genes involved in ethylene synthesis and signalling (ACS, ACO) was affected by TBB. This appears to contradict earlier studies invoking Ca2+ as an effector of transcript levels of both ACS and ACO (Petruzzelli et al., 2003). However, the latter study was carried out over a much longer time scale of 8 h and investigated the effect of Ca2+ in combination with the presence of ethylene. By contrast, this study points to a minor role of Ca2+ during the initial response phase to OGs, during which ethylene is likely to be synthesized.
These results suggest that, in the presence of TBB, OGs are able to bind to their putative receptors and that a substantial part of the OG signal transduction network remains active. However, the absence of a Ca2+ signal results in the inactivation of a large part of the ensuing jasmonate-based signalling. The possible role of Ca2+ in the induction of jasmonate accumulation in response to OGs was previously highlighted in a study by Hu et al. (2003) who, by exposing ginseng cells to LaCl3 and ruthenium red, showed that pretreatment with these Ca2+ channel inhibitors largely blocked OG-induced jasmonate biosynthesis.
One of the crucial junctions between the jasmonate and ethylene pathways is formed by transcription factors of the ethylene response factor (ERF) family. At this junction, the jasmonate and ethylene pathways are believed to converge (Fig. 3) and transcriptional activation of ERF1 is a key element in pathogen response signal integration and the regulation of the defence genes (Lorenzo et al., 2000). The data show (Table 2) that up-regulation of ERF1, and several other ERFs, occurs irrespective of Ca2+ signal inhibitors. Thus, transcriptional up-regulation of ERF1 per se does not appear to require the jasmonate pathway, in contrast to earlier speculation (Lorenzo et al., 2003).
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TBB is highly specific in its inhibitory action against the casein kinase CK2. Abolition by TBB of the OG-induced Ca2+ transient within seconds of the addition of OGs implies that a phosphorylation event forms part of the initial stages of the signalling pathway, possibly soon after binding of OGs with their receptor. Phosphorylation might directly impact on Ca2+ signalling, for example, through activation of Ca2+ channels. The activity of several animal Ca2+ channels has been shown to be increased by phosphorylation and, recently, Kimura and Kubo (2003) reported that the ß subunit of a squid plasma membrane Ca2+ channel contains a putative CK2 phosphorylation site that leads to channel activation when phosphorylated.
In the 5' upstream regions of genes that were significantly changed in transcript levels, both known and potentially new promoter cis-elements were identified. Several of the identified patterns contain poly-A stretches typical of repetitive, low complexity, sequences and therefore are unlikely to constitute genuine regulatory motifs. Furthermore, none of the previously described motifs could be correlated with TBB-sensitivity and hence with Ca2+ dependence. However, putative new elements were identified that only occurred in the TBB-sensitive categories and hence might form transcriptional regulatory domains requiring upstream Ca2+ signalling events. At this stage it can only be speculated about the precise role of such motifs, but some of them may be targets of Ca2+-dependent, jasmonate-regulated transcription factors. Direct interaction of transcription factors with calcium/calmodulin comprises another potential mechanism of Ca2+-dependent gene activation.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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Arabidopsis seeds expressing cytoplasmic aequorin were kindly provided by Dr Marc Knight (University of Oxford, UK). We would also like to thank Dr David Ashford (University of York, UK) for his expertise and help in the production of oligogalacturonides, Dr Chiara Romualdi (University of Padua, Italy) for help with statistical analysis of data, and Professor Flavio Meggio for kindly providing the protein kinase inhibitor TBB. Financial support was provided by an award of the Koerber Foundation.
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