Email updates

Keep up to date with the latest news and content from BMC Plant Biology and BioMed Central.

Open Access Highly Accessed Research article

Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis

Michael Wrzaczek1, Mikael Brosché1, Jarkko Salojärvi1, Saijaliisa Kangasjärvi2, Niina Idänheimo1, Sophia Mersmann3, Silke Robatzek34, Stanisław Karpiński5, Barbara Karpińska6 and Jaakko Kangasjärvi1*

Author Affiliations

1 Plant Biology Division, Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland

2 Department of Biology, University of Turku, FI-20014 Turku, Finland

3 Max-Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, DE-50829 Cologne, Germany

4 The Sainsbury Laboratories, Norwich Research Park, Colney Lane, NR4 7UH, Norwich, UK

5 Department of Plant Genetics, Breeding and Plant Biotechnology, Warsaw University of Life Sciences, Nowoursynowska st. 159, PL 02-776, Warsaw, Poland

6 Department of Life Sciences, Södertörn University College, SE-141 89 Huddinge, Sweden

For all author emails, please log on.

BMC Plant Biology 2010, 10:95  doi:10.1186/1471-2229-10-95


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2229/10/95


Received:14 October 2009
Accepted:25 May 2010
Published:25 May 2010

© 2010 Wrzaczek et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Plant Receptor-like/Pelle kinases (RLK) are a group of conserved signalling components that regulate developmental programs and responses to biotic and abiotic stresses. One of the largest RLK groups is formed by the Domain of Unknown Function 26 (DUF26) RLKs, also called Cysteine-rich Receptor-like Kinases (CRKs), which have been suggested to play important roles in the regulation of pathogen defence and programmed cell death. Despite the vast number of RLKs present in plants, however, only a few of them have been functionally characterized.

Results

We examined the transcriptional regulation of all Arabidopsis CRKs by ozone (O3), high light and pathogen/elicitor treatment - conditions known to induce the production of reactive oxygen species (ROS) in various subcellular compartments. Several CRKs were transcriptionally induced by exposure to O3 but not by light stress. O3 induces an extracellular oxidative burst, whilst light stress leads to ROS production in chloroplasts. Analysis of publicly available microarray data revealed that the transcriptional responses of the CRKs to O3 were very similar to responses to microbes or pathogen-associated molecular patterns (PAMPs). Several mutants altered in hormone biosynthesis or signalling showed changes in basal and O3-induced transcriptional responses.

Conclusions

Combining expression analysis from multiple treatments with mutants altered in hormone biosynthesis or signalling suggest a model in which O3 and salicylic acid (SA) activate separate signaling pathways that exhibit negative crosstalk. Although O3 is classified as an abiotic stress to plants, transcriptional profiling of CRKs showed strong similarities between the O3 and biotic stress responses.

Background

Receptor-like/Pelle kinases (RLKs) are important components in the regulation of plant development, hormone signalling, abiotic, and biotic stress responses in plants. RLKs are serine-threonine protein kinases that typically contain a signal peptide, a variable extracellular domain, a transmembrane region, and a conserved intracellular protein kinase domain. The extracellular ligand-binding domain perceives signals and is commonly used to classify RLKs into distinct subgroups [1]. The RLKs are one of the largest gene families in Arabidopsis with more than 600 members, [1-4], but only relatively few of them, mostly leucine-rich repeat RLKs (LRR-RLK), have been functionally characterized. CLAVATA1, a LRR-RLK, binds the small extracellular protein CLAVATA3 to regulate meristem proliferation [5]. FERONIA (a member of a previously uncharacterized group of RLKs) is central to the regulation of male-female interactions during pollen tube reception in Arabidopsis [6] and in Brassica the S-locus Receptor Kinase and its ligand are critical determinants of self-incompatibility [7,8]. In Arabidopsis, ERECTA (a LRR-RLK) is a multifaceted regulator of development and physiological processes as well as environmental responses [9]. BRASSINOSTEROID INSENSITIVE 1 (BRI1, a LRR-RLK) binds the plant hormone brassinosteroid and dimerizes with BRI1-ASSOCIATED RECEPTOR KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) [10,11]. BAK1 also inducibly dimerizes with the RLK FLAGELLIN SENSITIVE 2 (FLS2, a LRR-RLK), which recognizes bacterial flagellin and is important in plant immunity [12,13]. Other RLKs contributing to pathogen recognition include EFR (the Arabidopsis receptor for EF-Tu) and rice Xa21 (a LRR-RLK), which recognizes a sulfonated peptide produced by the pathogen Xanthomonas oryzae pv. oryzae [14-18].

The DUF26 (Domain of Unknown Function 26; PFAM domain PF01657) RLKs, also known as Cysteine-rich RLKs (CRKs), form a large subgroup of the RLK family with more than 40 members [1,19]. The extracellular region of the protein contains two copies of the DUF26 domain which has four conserved cysteines (three of them form the motif C-8X-C-2X-C) that may form disulphide bridges as potential targets for thiol redox regulation. The CRKs are transcriptionally induced by oxidative stress, pathogen attack and application of salicylic acid (SA) [19-22]. Accordingly several members of the CRK subgroup of RLKs are involved in the regulation defence reactions and cell death in Arabidopsis leaves. Constitutive over-expression of CRK5 led to increased resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but also to enhanced growth of the plant leaves [22]. Over-expression of CRK4, CRK5, CRK19 and CRK20 by a chemically inducible promoter, on the other hand, caused cell death [19,22]. Genetic analysis suggested that CRK5 regulated cell death independently of SA [22]. Conversely the enhanced resistance to Pseudomonas upon overexpression of CRK13 required increased SA levels [23].

Reactive oxygen species (ROS) have been established as important signalling molecules for inter- and intracellular communication in plants, animals and yeast [24-26]. ROS are produced in strictly defined locations in reponse to specific stimuli [25]. Pathogen infection rapidly induces an extracellular oxidative burst while light stress and specific chemicals, including paraquat and norflurazon, induce ROS production in the chloroplast [27-29]. Plant cells can differentiate between the type and localization of ROS resulting in very specific responses. Furthermore, ROS production in specific cellular compartments can have impact on ROS generation and signalling in other locations [30,31]. This crosstalk is likely accomplished through interplay between separate signalling pathways rather than direct interaction of the ROS molecules themselves [30,31]. However, the molecular components and mechanisms involved are still poorly defined [31,32]. In addition, it is unknown how ROS are sensed and how specificity in ROS signalling is achieved. The gaseous molecule ozone (O3) induces a burst of ROS in the apoplast similar to the oxidative burst in plant-pathogen interactions [24]. Other similarities between O3 and pathogen infection include the production of SA and ethylene (ET) [24]. O3 is a convenient system to experimentally address the effects of apoplastic ROS since the plant is not exposed to other effector proteins or toxins which might induce defence responses. O3 permits the study of the apoplastic oxidative burst undisturbed by manual manipulation of the plant material.

Plant hormones are a group of unrelated small compounds which are central to signalling during environmental adaptation and developmental regulation [33,34]. SA, jasmonic acid (JA) and ET are viewed as the main hormonal determinants of plant pathogen defence [35,36]. Abscisic acid (ABA) modulates plant defence and is a negative regulator of SA responses [37]. In addition, ABA is a key regulator of the high light response [38]. The interaction of hormone and ROS signalling is well documented. ROS can induce cell death in a SA-dependent and independent manner [24]. Cell death and ROS induce ET synthesis, which feeds into a positive forward amplification loop enhancing ROS production [39]. ROS-induced JA is critical in limiting cell death [24]. Thus, the successful outcome of a given response is not determined by one hormone, but is achieved through balance, interaction and constant recalibration of different plant hormones.

Despite extensive research on ROS signalling, the exact components mediating ROS signalling, ROS sensing, and perception in particular are still unknown. Here we have analysed transcriptional regulation and the involvement of hormonal signalling in regulating the expression of the whole Arabidopsis CRK gene subfamily by ROS. The effects of ROS production in different subcellular compartments was analysed by using O3- and light stress treated plant material and publicly available microarray data. We show that O3-induced transcriptional responses are blocked in the defense, no death 1 (dnd1) mutant, and they are altered in hormone biosynthesis or signalling mutants. Collectively this reveals alternate pathways in the regulation of ROS responses.

Results

CRK transcriptional response to O3

Several groups of RLKs are transcriptionally regulated in response to biotic stresses [40]. We identified several CRKs which were differentially regulated by O3 (MB and JK unpublished microarray data). These results suggest a strong transcriptional regulation of the CRKs during stress responses. Therefore we chose to investigate further the transcriptional regulation of the whole CRK subfamily by ROS.

According to Shiu and Bleecker [1], Chen et al. [19], and our analysis (see table 1 for nomenclature and reference), the CRK subfamily consists of 44 members. Previously two additional genes have been included, but At4g11500 (DUF26 44) was classified as a pseudogene in the current version of the Arabidopsis genome (TAIR9; http://www.arabidopsis.org webcite[41]) and At4g23170 (CRK9) contains no identifiable extracellular domain, signal peptide or complete kinase domain; thus both genes were excluded from the analysis.

Table 1. Nomenclature of the CRKs/DUF26 RLKs.

We analysed the transcriptional responses of all the 44 CRKs to extracellular ROS produced by O3 by quantitative real-time RT-PCR (qPCR). Out of the 44 CRKs, 25 (nine with statistical significance FDR [False Discovery Rate]-corrected p-value ≤ 0.1; additional file 1) showed more than two-fold higher mRNA abundance after 1-hour exposure to O3 (Figure 1). After a 6-hour O3 exposure followed by a 2-hour recovery period, 26 CRKs exhibited a more than two-fold increase in expression (eight with statistical significance FDR-corrected p-value ≤ 0.1; additional file 1). Only CRK22, CRK30, CRK32, CRK33 and CRK46 showed decreased expression in response to O3-treatment. In order to analyze if transcriptional regulation after exposure to O3 was a feature of a single subset of the CRKs, the protein sequence of the kinase domain of all CRKs was aligned to construct a Neighbour-joining tree representing the relations between the members of the CRK group of RLKs (Figure 2). CRKs that were transcriptionally regulated in response to O3 are high-lighted. O3-regulated genes were distributed across the tree instead of forming a unique branch. However, closely related genes showed a tendency to share similar O3 expression patterns.

Additional file 1. Lower and upper percentiles and p-values. The raw normalized cycle differences (ΔΔCt) for all experiments, their average, standard deviation, geometric mean, lower and upper percentile and the Benjamini-Hochberg False Discovery Rate-corrected p-value for all experiments is shown in the Excel File. Each Excel worksheet represents data for a Figure showing qPCR data.

Format: XLS Size: 235KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

thumbnailFigure 1. Transcriptional regulation of the CRKs in response to O3. Expression of all members of the CRK group of plant RLKs was analyzed by quantitative real-time RT-PCR (qPCR) in Col-0 plants exposed to 250 ppb O3 for 6 h. Samples were harvested at 1 or 8 h (6 h followed by 2 h recovery under clean air conditions) after the onset of the O3 treatment. Transcript levels were calculated by comparison of O3-exposed plants with corresponding control plants grown under clean air conditions harvested in parallel with the O3-treated plants. An expression level of one indicates no change in expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase in expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistical significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

thumbnailFigure 2. Phylogenetic tree of the CRK kinase domains indicates that O3 regulation is distributed throughout group. The kinase domains of all CRKs were aligned using ClustalW2 and a Neighbour-joining tree was constructed using MEGA4 [84]. DUF26 44 (At4g11500) and CRK9 (At4g23170) were not included in the analysis. Genes with increased expression by O3 treatment are indicated in red and genes with decreased expression in green (statistically significant changes are indicated by an asterisk).

CRK transcriptional response to light stress

To determine the effects of light stress-induced ROS production, we monitored the expression of ASCORBATE PEROXIDASE 2 (APX2), encoding a ROS scavenger and established marker for light-induced ROS production [42]. APX2 was strongly induced after 1- and 2-hour exposure to light stress conditions (Figure 3). In contrast to O3 (Figure 1), light stress led to rapid transcriptional repression of several CRKs (Figure 3). Twenty CRKs were transcriptionally repressed while only eight exhibited increased expression. However, the light-dependent regulation of the CRKs was not statistically significant. The lack of transcriptional induction in response to light stress corresponds to results from Lehti-Shiu et al. [40], who reported that the CRKs were transcriptionally strongly induced in response to biotic stimuli but the expression level decreased in response to abiotic stress (including heat, cold, drought and salt). Of the abiotic treatments, only UV-B, osmotic stress and wounding resulted in increased expression of CRKs [40].

thumbnailFigure 3. Transcriptional downregulation of CRKs in response to light stress. Expression of APX2 (a marker for light stress) and CRKs was analyzed by qPCR in plants after 1 h and 2 h exposure to light stress conditions and after 4 h light stress followed by 4 hours recovery at normal growth light conditions. Transcript levels were calculated by comparison of light stress-treated plants with corresponding control plants grown under normal light conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated twice; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistical significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

CRK transcriptional response to PAMPs is similar to the O3 response

To more broadly address transcriptional regulation of the CRKs, we analyzed and compared their expression profiles from publicly available Affymetrix chip data. Raw data files were obtained from several databases (see material and methods) and RMA (Robust Multi-Array Average) normalized. To take the sample variation into account, parametric bootstrapping combined with Bayesian hierarchical clustering [43] was applied. This results in a numerical measure of similarity between treatments and genes, which can be clustered hierarchically (Figure 4; for a related application, see [44]). The meta-analysis of the publicly available O3 microarray data revealed high overlap with our qPCR data; all eight genes with more than 3-fold increased expression in the publicly available array data exhibited increased expression in our qPCR analysis. Treatment with norflurazon (which increases singlet oxygen [1O2] in the chloroplast causing excess ROS production) led to decreased expression of four CRKs. Norflurazon blocks carotenoid biosynthesis and thus removes this quencher of the triplet chlorophyll and 1O2. Paraquat leads to superoxide production in the chloroplast by transferring electrons from photosystem I to oxygen. The is subsequently dismutated to H2O2. Paraquat had no effect on CRK expression with the exception of the latest time point tested (24 hr), whereupon five CRKs exhibited increased expression; four of which were also regulated in response to O3. However, at this time point paraquat had most likely induced cell death. H2O2 treatment selectively led to increased expression of a few CRKs which also displayed increased expression by O3. Rotenone (an inhibitor of mitochondrial electron transport causing elevated ROS production in mitochondria) had little impact on CRK expression; only CRK3 showed increased expression levels. Thus, the CRK expression profile triggered by O3 was not related to expression profiles established by other ROS treatments. Instead, the O3-triggered CRK expression profile clustered together with that provoked by several biotic and PAMP treatments, including Blumeria graminis var. hordei (Bgh), harpin Z (HrpZ), and the flagellin elicitor-active epitope flg22 (Figure 4).

thumbnailFigure 4. Bayesian hierarchical clustering of the CRKs in abiotic and biotic stress experiments. Biotic and abiotic stress data sets were down loaded from public databases and included O3, norflurazon, paraquat, BTH (benzothiadiazole S-methylester), various elicitors and pathogens (see materials and methods for complete details). Red and green indicate increased or decreased expression compared to untreated plants, respectively. The intensity of the colours is proportional to the absolute value of the fold difference.

Our qPCR analysis confirmed the changes caused by flg22 in the expression profile of the CRKs obtained from publicly available microarray data (Additional file 2 and Figure 4). Treatments with benzothiadiazole S-methylester (BTH; an active SA analog) resulted in two-fold or higher up-regulation of 12 CRKs, some of which also exhibited elevated expression in response to O3. Interestingly, in the non-expressor of pathogenesis-related genes 1 (npr1) mutant these genes were not regulated by BTH treatment (Figure 4), indicating that SA regulation of these genes was dependent on NPR1-mediated signalling. Application of methyl-jasmonate (MeJA) did not cause any major changes in CRK expression (Figure 4), whilst ABA treatment resulted in decreased expression of CRK25, CRK30, CRK28, CRK29, CRK19, CRK21 and CRK22 at late time points. Overall, the CRK expression profile in response to BTH clustered together with that triggered by O3, pathogen and PAMP treatments; whereas CRK transcriptional regulation upon ABA application clustered together with paraquat, norflurazon, rotenone and MeJA treatments (Figure 4).

Additional file 2. Transcriptional regulation of the CRKs in response to flg22. 14-day old Arabidopsis Col-0 were treated with 100 nM flg22 and samples taken after 30 and 60 minutes (water-treated control samples have been harvested at the same time points in parallel). Expression of several CRKs was analyzed by qPCR. Transcript levels were calculated by comparison with the corresponding control plants. An expression level of one indicates no change in expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase in expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green.

Format: PNG Size: 149KB Download fileOpen Data

Taken together, these results demonstrate that the CRK expression profile in response to O3 is not related to treatments which mediate ROS production in the chloroplast or the mitochondria. However, there is a substantial overlap between the transcriptional responses to O3 and pathogen infection/PAMP perception, which may be a result of apoplastic ROS commonly generated by all these stimuli.

CRKs display different expression in hormone mutants

Altered transcriptional regulation of several CRKs has previously been shown following external application of the plant hormone SA or its active analog BTH (Figure 4 and [19]). In order to address the impact of hormone signalling on transcriptional regulation of CRKs, we used several mutants impaired in hormone biosynthesis and/or signalling. The salicylic acid induction deficient 2 (sid2) mutant is deficient in SA biosynthesis (due to a mutation in the SA biosynthesis gene ISOCHORISMATE SYNTHASE 1 [ICS1]), whilst npr1 is impaired in SA signalling. The dnd1 mutant fails to produce a hypersensitive response (HR), but has functional effector-triggered immunity, constitutive systemic resistance and accumulates elevated SA levels [45-47]. The ethylene insensitive 2 (ein2) mutant is deficient in ET signalling, and the fatty acid desaturase 3/7/8 (fad3/7/8) mutant is deficient in JA biosynthesis. We compared the transcript abundance of CRKs in these mutants to Col-0 wild type plants using qPCR. The obtained Actin-2-normalized threshhold cycle values (Ct) were compared between Col-0 wild type and the mutants. Several CRKs showed lower expression in sid2 and npr1 (Figure 5A). CRK29 displayed higher expression in sid2 and ten CRKs (three with statistical significance FDR-corrected p-value ≤ 0.1) exhibited higher expression in npr1. In the ein2 and fad3/7/8 mutants, for nine and twelve CRKs, respectively, expression levels were elevated as compared to wild type plants. Only CRK7 and CRK8 showed lower expression in ein2. Along with several other defects, dnd1 exhibits constitutive SA responses [48], which might be the cause for the increased transcript levels of 15 CRKs in dnd1 signalling -however, other regulatory mechanisms cannot be ruled out due to the pleiotropic nature of the mutant [48]. Expression of some CRKs was unaltered or displayed only subtle changes in the sid2 mutant, but was elevated in npr1, ein2, fad3/7/8 and dnd1 mutants (CRK6, CRK23, CRK26, CRK36, and CRK45). Interaction between hormone signalling pathways is an established phenomenon [24,37], and the CRKs above exemplify that altering the balance of SA, JA or ET response leads to altered gene expression.

thumbnailFigure 5. Expression of CRKs is changed in hormone mutants. The expression of all CRKs was analyzed by qPCR in the SA mutants sid2 and npr1, the ET mutant ein2, the JA mutant fad3/7/8 and the cell death mutant dnd1 by qPCR and compared to Col-0 under two different growth conditions. (A) Weiss chamber conditions. (B) Phytotron. Transcript levels were calculated by comparison between mutants and Col-0 grown under control conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. ND - The dnd1 mutant did not grow in the phytotron. Fold-change is shown for the geometric mean of all biological repeats (n = 4). Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

To test the robustness of gene expression in this set of hormone mutants, we compared two different growth conditions. These differed in photoperiod, light composition and intensity, soil composition and humidity (see Materials and Methods for a detailed description of the differences in the growth conditions), subsequently referred to as Weiss chamber (Figure 5A) and Phytotron (Figure 5B). Notably, the dnd1 mutant did not grow under Phytotron conditions. The higher transcript abundance of CRKs in ein2 and fad3/7/8 observed in plants grown under Weiss chamber growth conditions was largely absent in plants grown under Phytotron growth conditions (Figure 5B). Moreover, the CRKs which showed higher gene expression in npr1 under Weiss chamber growth conditions, were unaltered (or had even reduced transcript levels) in the Phytotron. Taken together, these results indicate that hormones play a major role in the transcriptional regulation of many CRKs. However, environmental growth conditions also have a large impact on the extent of this regulation especially in soil grown plants [49,50].

O3-response of the CRKs in hormone mutants

To further study the role of SA, ET and JA in ROS signalling, wild type and the sid2, npr1, dnd1, ein2 and fad3/7/8 mutants were exposed to O3. A subset of 23 O3-induced and one O3-repressed CRKs were selected for expression analysis in the mutant backgrounds by qPCR (Figure 6). Most O3-induced CRKs exhibited even higher expression levels in sid2 and npr1 as compared to wild type, with the exception of CRK10, CRK11, CRK20 and CRK29. In ein2, the magnitude of CRK induction was reduced. In the JA-deficient fad3/7/8 mutant, the increased expression of CRKs in response to O3 was in several cases reduced or even absent as compared to wild type plants. Remarkably, O3-triggered increase in expression of CRKs was absent in dnd1 (Figure 6). In summary, these results suggest that the plant hormones SA, JA and ET play central roles in the regulation of the expression of the CRK subfamily, both under control conditions (clean air), as well as in response to O3.

thumbnailFigure 6. O3-regulation of CRKs is different in hormone mutants. The expression of 24 O3-regulated CRKs was analyzed by qPCR in Col-0 and sid2, npr1, dnd1, ein2 and fad3/7/8 exposed to 250 ppb O3 for 6 h. Samples were harvested at 1 or 8 h (6 h plus 2 h recovery under clean air conditions) after the onset of the O3 treatment. Transcript levels for Col-0 or each mutant line were calculated by comparison of O3-exposed plants with corresponding control plants of the same line grown under clean air conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

To expand the model for O3 regulated gene expression, we tested several other O3 inducible marker genes. These genes were selected to represent "classical" marker genes for SA (including PATHOGENESIS-RELATED GENE 1 [PR-1] and PATHOGENESIS-RELATED GENE 2 [PR-2] and JA/ET (PLANT DEFENSIN 1.2 [PDF1.2]). In addition we selected genes based on our previous O3 microarray data (SENESCENCE-ASSOCIATED GENE 21 [SAG21] [51]), and genes which have previously been described as JA-regulated (MONODEHYDROASCORBATE REDUCTASE [MDHAR] [52]) or SA- and NPR1-regulated (LECTIN-LIKE PROTEIN [LLP] At5g03350 [53]). The overall regulation of the marker genes was obtained by clustering them in response to biotic and abiotic stress and hormone treatments (Figure 7A). Most of the genes were regulated in response to BTH, biotic stress treatment and O3, and the MDHAR gene was confirmed as a JA marker gene, as previously reported [52]. However, there was a lack of overall "specificity" in marker gene expression, i.e., several hormones or stresses were altering their expression. The marker genes were next tested with qPCR in the same O3 samples used for CRK expression. The genes were strongly induced in Col-0 wild type plants and in most mutants. However, in dnd1 the O3-induced signalling pathway(s) was evidently blocked since O3-induced gene expression was not observed or it was severely reduced. Only PATHOGENESIS-RELATED GENE 5 (PR-5) was weakly induced in dnd1 at the later time point. The classical SA marker genes PR-1 and PR-2 had reduced O3-induced increased expression in sid2 and npr1, indicating a role for SA signalling in response to O3. The loss of O3 induction of MDHAR in fad3/7/8 confirmed the importance of JA in regulation of this gene.

thumbnailFigure 7. Clustering and qPCR analysis of the marker genes. (A) The expression of eight O3-inducible genes and the qPCR normalization gene Actin-2 were analyzed in public array data from biotic and abiotic stress and hormone treatments. Red and green indicate increased or decreased expression compared to untreated plants, respectively. The intensity of the colours is proportional to the absolute value of the fold difference. (B) Markers genes for O3 responses were analyzed by qPCR in Col-0 and sid2, npr1, dnd1, ein2 and fad3/7/8 exposed to 250 ppb O3 for 6 h. Samples were harvested at 1 or 8 h (6 h plus 2 h recovery under clean air conditions) after the onset of the O3 treatment. Transcript levels were calculated by comparison of O3-exposed plants with corresponding control plants grown under clean air conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Light stress response of the CRKs in hormone mutants

To elucidate the role of SA, JA and ET in the regulation of CRK expression in response to light stress, wild type and the sid2, npr1, ein2 and fad3/7/8 mutants were exposed to light stress and the subset of O3-regulated CRKs was analyzed by qPCR. The transcriptional repression observed in response to light stress (Figure 3) for a majority of CRK family members was even more pronounced for some CRKs in sid2 (Figure 8). Interestingly, several CRKs were specifically transcriptionally induced by light stress in the ein2 mutant. In fad3/7/8, most CRKs exhibited a transient decrease in gene expression at early time points. However, statistical significance was overall low for the light-dependent regulation of the CRKs in the hormone signalling and biosynthesis mutants (Additional file 1).

thumbnailFigure 8. Light stress response in hormone mutants. The expression of 24 O3-inducible CRKs was analyzed by qPCR in Col-0 and sid2, npr1, ein2 and fad3/7/8 after 1 h and 2 h exposure to light stress conditions, and after 4 h light stress followed by 4 h recovery at normal growth light conditions. Transcript levels were calculated by comparison of light stress-treated plants with the corresponding control plants grown under normal light conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase in expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated twice; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

CRK promoter analysis

Gene expression is regulated by transcription factors and the promoter elements they bind to. The 500 base pair (bp) and 1000 bp upstream promoter regions of the CRKs were inspected for significantly enriched promoter elements based on a list of verified Arabidopsis promoter elements (http://arabidopsis.med.ohio-state.edu/AtcisDB/bindingsites.html webcite[54]). The CRKs were divided into three groups ("CRKs all", "CRKs O3 up" - O3 increased expression and "CRKs O3 down" - O3 decreased expression) and searched for significant accumulation of single promoter elements or a combination of promoter elements. Statistical significance was measured with the Fisher exact test using false discovery rate correction [55]. The enrichment was calculated separately for the motifs in both forward and reverse orientations. No elements were enriched in the 1000 bp region for any of the groups or in the 500 bp region of O3 down genes. One element, the W-box, a target for WRKY transcription factors frequently found in the promoters of SA-regulated genes [56], was significantly overrepresented as a single motif in the group of "CRKs all" and "CRKs O3 up" in the 500 bp region (Table 2 and Additional file 3). Interestingly, several pairs of promoter elements were present with high statistical significance in the 500 bp region for the "CRKs O3 up" and "CRKs all" groups. Since these were mostly the same for both groups and had high statistical significance for the all group, this indicated that they were probably not responsible for the O3-regulation of these genes. The W-box was the only element enriched as a single motif but also present in most pairs of promoter elements. This indicated that the W-box, alone or in combination with other elements, could be a target for the SA and/or pathogen regulation of CRKs.

Additional file 3. List of CRKs for promoter motifs in table 2. This file lists the AGI codes for the CRKs containing the promoter motif combinations shown in table 2.

Format: XLS Size: 97KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

Table 2. Motifs overrepresented in the promoters of the CRK family.

Discussion

The RLK family is one of the largest gene families in the Arabidopsis thaliana genome. Several RLKs have previously been described to be involved in plant-microbe interactions [14,15,57-59] and abiotic stress [60,61]. Based on statistical analysis of gene expression data, RLKs in general, as well as the CRK subfamily, are more likely to have altered expression in response to abiotic and biotic stress than other Arabidopsis genes [40,62]. We analyzed the expression profile of the CRKs in detail using qPCR and array analysis under various stresses, growth conditions, and in different genetic backgrounds to obtain a better understanding of the signalling pathways leading to transcriptional regulation of the CRKs and to elucidate the role of apoplastic ROS in stress signalling.

The use of ROS as signalling molecules is a common feature of many stress responses [25]. Pathogen attack and perception of PAMPs are often associated with an oxidative burst in the apoplast [63]. Similarly, a hallmark of the early O3 response is the generation of an oxidative burst in the apoplast [64]. ROS are also produced in other subcellular compartments, including the chloroplast, where light stress or treatments with the herbicides paraquat or norflurazon elicit elevated ROS production. In addition, crosstalk between pathways elicited by apoplastic ROS and chloroplast-derived ROS is important for the regulation of cell death [32]. The transcriptional response to apoplastic ROS, e.g. induced by O3, is strikingly different from chloroplast-derived ROS, e.g., induced by paraquat [30]. To further dissect the role of apoplastic ROS, we clustered several treatments triggering ROS production in distinct subcellular compartments together with various biotic stress experiments. Our results showed that the CRK expression profile upon O3 exposure was most similar to those stimulated by PAMP perception (flg22 and HrpZ) and pathogen infection (Bgh) (Figure 4). By contrast, treatments, which increased ROS levels in the chloroplast (norflurazon and paraquat) or mitochondria (rotenone; which might also lead to ROS production in the chloroplast [65]) either had no effect on CRK gene expression or resulted in down-regulation. These results show that transcriptional induction of the CRKs can be triggered by apoplastic ROS, whereas chloroplastic ROS mainly lead to decreased expression. Furthermore, cluster analysis separated the effects of plant hormones: BTH (SA analog) caused a similar expression profile as O3 and PAMP treatments, whereas CRK expression in response to ABA and MeJA was related to norflurazon and paraquat treatments.

To extend the microarray meta-analysis, transcript accumulation of the CRK subfamily was monitored in response to O3 and light stress by qPCR. Out of 44 CRKs, 32 showed increased expression after exposure to O3 at both time points while five members exhibited decreased expression. Light stress treatment led to a decrease in expression of the majority of the CRKs. Thus, in agreement with the results from array analysis, ROS production in different cellular compartments produces strikingly different transcriptional profiles on the CRK gene subfamily.

To further dissect the O3 response, mutants deficient in biosynthesis, perception and signalling of SA (sid2, npr1), JA (fad3/7/8) and ET (ein2) were exposed to O3 and the expression of a subset of CRKs was analyzed by qPCR. The O3-induced increase in transcript levels of the CRKs was higher in sid2 and npr1 implying that SA acts as a negative regulator of the ROS signalling pathway. The O3-mediated transcriptional induction of CRKs was almost abolished in fad3/7/8 and attenuated in ein2, suggesting that JA, and to a lesser extent ET are required for the proper transcriptional induction of CRKs in response to O3. This role for SA, JA and ET in O3 signalling has been previously proposed based on the results from cDNA macroarray analysis [66]. The effect of light stress on the CRK expression in various mutant backgrounds was very different compared to the effect of the O3 response. Whereas ET acts as positive regulator of CRK expression in the O3 response, it appears to be a negative regulator in light stress since several CRKs displayed light stress-induced expression only in the ein2 mutant (Figure 8). Under light stress conditions, the decreased expression of CRKs seen in wild type was even more pronounced in the SA mutants sid2 and npr1 and the JA mutant fad3/7/8.

DND1 encodes CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2) which transports Ca2+ into the cell and regulates nitric oxide production [67]. The complete lack of an effect of O3 on CRK and marker gene expression in dnd1 suggests an important role for CNGC2 in the O3 response pathway, possibly by regulating Ca2+ levels (Figure 6 and 7B). Previous studies have shown that O3 rapidly invokes Ca2+ transients [68,69] and blocking of Ca2+ transport can prevent ROS-induced cell death [70]. The dnd1 mutant also has several pleiotropic phenotypes which include elevated SA levels and constitutive defence responses [47]. Consequently, the lack of O3 response in dnd1 could be due to "dominance" of SA signaling over the ROS signalling pathway, and O3 would have no effect when the SA pathway is fully stimulated. Previous reports have shown that several members of the CRK subfamily were transcriptionally induced through an external application of SA [19] or BTH (Figure 4). The response of CRKs to BTH was completely blocked in npr1, indicating that the SA pathway for regulating CRKs requires NPR1.

Intriguingly, different growth conditions had a strong impact on the expression of CRKs in various mutants. Several CRKs were expressed to higher levels in ein2 and fad3/7/8 in Weiss chamber-grown plants compared to Phytotron-grown plants. In contrast, the decreased expression of several CRKs in sid2 and npr1 was similar between two different growth conditions (Weiss chamber and Phytotron, Figure 5). A strong effect of environmental conditions on mutant phenotypes, transcript profiles and other parameters are well known and a common problem when comparing results from different laboratories [71]. There could be several reasons for the differences in the expression levels of the CRKs between the Weiss chambers and the Phytotron growth conditions. Plants were tested at slightly different ages and grown in different soil (see materials and methods section). Illumination in the Weiss chambers was provided using fluorescent lamps while in lighting in the Phytotron was using metal halide lamps with different light spectra. Notably, the CRKs are responsive to UV-B [40]. This suggests that light conditions could have an effect on the expression profile of this RLK family. Another reason for this variation of gene expression could be that under control conditions most CRKs were expressed at very low levels; consequently, a minor perturbation either by genetic mutation or growth condition could lead to altered expression. Thus, expression of CRKs is very sensitive to the surrounding environment. Similar observations have been reported for the expression of the classical PDF1.2 marker gene [49,50]. This gene has long been used to exemplify co-regulation by JA/ET. However, PDF1.2 is only regulated by both hormones when plants are grown in vitro [49]. When plants are grown in soil, either hormone alone (JA or ET) is sufficient to induce expression. Thus, growth in soil is able to induce or prime defence signalling pathways.

Conclusions

Based on the CRK expression patterns and integrating current knowledge of ROS signalling, PAMP perception and light responses [25,26,38,72], we propose a model for the regulation of increased expression of the CRKs (Figure 9): O3 induces ROS production in the apoplast which is perceived by putative "ROS receptors" (or by other mechanisms) amplified by PLANT RESPIRATORY BURST OXIDASE HOMOLOG (RBOH)-mediated production, thus leading to activation of DND1/CNGC2. This activates further down-stream signalling events where JA and to a lesser extent ET act as positive regulators, and SA and NPR1 as negative regulators of CRK expression. Eventually, the signal reaches the nucleus where transcription factors bind to a "ROS" promoter element and activate transcription. In parallel, the genes are also regulated through a SA (synthesized by ICS1) and NPR1-dependent pathway converging on the W-box promoter element. Microbes and PAMPs could activate both pathways at different timing; a rapid pathway would act through a RBOH mediated ROS production and use the "ROS pathway", while a later "SA pathway" requires increased SA biosynthesis and NPR1. Further interconnections between the pathways are provided by the primary ET transcription factors ETHYLENE INSENSITIVE 3 (EIN3) and ETHYLENE INSENSITIVE 3-LIKE (EIL1) which repress SID2/ICS1 expression and thus decrease SA levels [73]. Light stress or chemical treatments that increase ROS in the chloroplast activate separate signalling pathway(s) mainly leading to repression of CRK expression, which could involve ABA and negative crosstalk with the SA pathway.

thumbnailFigure 9. ROS, elicitor and hormone regulation of O3-induced CRKs. O3 enters the leaves through the stomata and immediately reacts with components of the cell wall to generate ROS. O3 and the ROS induce an active production of ROS in the apoplast which is at least partly depending on membrane bound NADPH oxidases (RBOH), which produce . Similar ROS production in the apoplast takes place after infection of a plant with a pathogen or treatments with pathogen derived elicitors (PAMPs). ROS is hypothetically perceived via a "ROS receptor" which could sense ROS directly via protein modification, or via sensing of modified apoplastic proteins or other molecules that react with ROS. The perception of ROS initiates down-stream signalling events. H2O2 is also able to cross the plasma membrane and enter the cells. Inside the cell, the signalling pathway is split into two pathways. In the ROS pathway DND1/CNGC2 mediates a required step of the signalling pathway and JA and ET act as positive regulators, and SA and NPR1 are negative regulators. In the SA pathway ROS or pathogens activate SA biosynthesis via ICS1; and NPR1 is a required component. Since NPR1 is a positive regulator of the SA pathway and a negative regulator of the ROS pathway this implies that the separate signalling pathway use different transcription factors and promoter elements to regulate CRK expression, although it might be possible that two different transcription factors could converge on the same promoter element. In addition the pleiotropic nature of the dnd1 mutant, including high SA-levels, could change the place of DND1/CNGC2 in the model - constitutive SA signalling in dnd1 may limit the possibility for O3 to activate the ROS pathway. Through the transcription factors EIN3 and EIL1 ET can repress SID2/ICS1 expression and SA levels. Increased ROS production in the chloroplast activates separate signalling pathway(s) leading to repression of CRK expression. One of these pathways could involve ABA and negative cross talk with the SA pathway.

Is it possible to separate the roles of chloroplastic and apoplastic ROS in the regulation of CRK expression? Chloroplast-derived ROS production is known to be involved in the regulation of cell death during pathogen infection and in response to abiotic stress [74,75]. Specific removal of chloroplastic ROS prevents pathogen-induced cell death but has no impact on defence gene expression [75]. Furthermore, chloroplastic 1O2 regulates cell death dependent on EXECUTER1 [31]. In comparison, apoplastic ROS might be involved with intra- and intercellular signalling [76]. Thus, apoplastic ROS would have a role in regulating defence gene expression and chloroplastic ROS in regulation of cell death. In addition, there is crosstalk between apoplastic ROS and chloroplast ROS; rapid ROS production in the chloroplast can be detected in response to O3 and blocking of ROS production in the chloroplast reduces O3-induced cell death [32,77]. Clearly, ROS regulation of defence signalling and/or cell death is very complex and several other regulatory components have been identified, including LESION SIMULATING DISEASE 1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4), which are also involved in acclimation to light stress [42,78]. The only known phenotypes for CRKs have been obtained by ectopic overexpression, which induces HR-like cell death independent or dependent on SA signalling (depending on the specific CRK) [22,23]. How this induction of cell death might be achieved is still unclear since transcriptional regulation of CRKs occurs in response to apoplastic rather than chloroplastic ROS. Some members of the RLK family might participate in a positive feed-forward loop to regulate ROS production, defence gene expression, cell death and hormone signalling. This regulatory loop might be deregulated after overexpression of the CRKs leading to the observed cell death phenotypes. However, this will require experimental verification in the future.

What is the role of CRKs in plants and why are they regulated by PAMPs and O3 treatment? The external domain of these RLKs could be the receptor for as yet uncharacterized PAMPs and they could be part of plant immune responses. An intriguing feature of the DUF26 domain is the presence of a conserved cysteine motif C-8X-C-2X-C. The configuration of cysteines is similar to the cysteine motif in the GRIM REAPER protein, which has been shown to be involved in the regulation of ROS induced cell death [79]. Despite the ubiquitous role of ROS as signalling molecules in plants, no direct receptor for ROS has been described. Since cysteines are sensitive to redox modifications, could the DUF26 domain act as sensor of ROS in the apoplast and be the putative ROS sensor as depicted in Figure 9?

Methods

Plant growth conditions and treatments

Weiss chamber growth conditions

For exposure to O3, Arabidopsis thaliana Col-0 or mutant plants were grown in a peat/vermiculite (1:1) mixture for 21 days in Weiss 1300 growth cabinets (photon flux density 250 μmol m-2 sec-1; tubular fluorescent lamps) under 12 hours day length (day: 23°C 70% relative humidity; night 18°C 90% relative humidity). Lights were switched on at 7 AM and off at 7 PM. O3 treatments were started at 9 AM. 21-day old plants were used and exposed to 250 parts per billion (ppb) O3 for 6 hours. Samples were harvested at the times indicated in the respective experiments after the onset of the O3 treatment. Samples were taken in parallel from O3 treated and clean air control plants and immediately shock-frozen in liquid nitrogen.

Phytotron growth conditions

For light stress treatments, plants were grown on a pre-fertilized garden soil/vermiculite (1:1) mixture for 28 days under 8 h/16 h light/dark at 22 or 20°C, respectively, and 50% humidity at a light intensity of 130 μmol m-2 sec-1 photon flux density (Metal halide lamps). For light stress treatment, plants were shifted to 1300 μmol m-2 sec-1 photon flux density for up to 4 hours. Subsequently, plants were returned to a light intensity of 130 μmol m-2 sec-1 photons. Controls were kept at 130 μmol photon flux density throughout the duration of the treatment and samples were taken in parallel with the light stress-treated plants. Samples were harvested at the times indicated in the respective experiments after the onset of the light stress treatment and immediately shock-frozen in liquid nitrogen.

For flg22 treatments, plants were grown on MS plates with Nitsch vitamins (MSN). After 7 days, seedlings were transferred to liquid MSN media and cultivated for 7 days. Before the flg22 treatment, fresh medium was added. After a 1 hour recovery period, the seedlings were treated with 100 nM flg22. Controls were treated with H2O. Samples were harvested at the times indicated in the respective experiments after the onset of the treatment and in parallel from corresponding controls and immediately shock-frozen in liquid nitrogen.

RNA extraction and qPCR analysis

RNA was isolated as described [79]. 5 μg total RNA was DNaseI treated (Fermentas) and used for cDNA synthesis with RevertAid Premium Reverse Transcriptase (Fermentas) and Ribolock RNase Inhibitor (Fermentas) according to manufacturers' instructions. The reaction was diluted to a final volume of 50 μl and 1 μl cDNA was used as template for PCR using LightCycler 480 SYBR Green I master mix (Roche Diagnostics) on a LightCycler 480 (Roche Diagnostics) in triplicate. Primer sequences and the primer amplification efficiency (Ex; determined according to manufacturers instructions) are available in additional file 4.

Additional file 4. Primer sequences for qPCR analysis. All primer sequences used for qPCR analysis in the manuscript plus the experimentally determined primer amplification efficiencies Ex are listed.

Format: XLS Size: 31KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

For the normalization of the data several genes were evaluated to select a suitable gene for normalization based on the method of Vandesompele et al. [80]. Actin-2 (At3g18780) was found to be stably expressed in control and ozone treated plants and was subsequently used for normalization. The raw Ct values were normalized to Actin-2 and used to compare the results from untreated control samples with treated samples using the 2-ΔΔCt method. The resulting normalized cycle differences were used to calculate the average (μ) and standard deviation (σ) of the biological repeats and the p-value (using SPSS) based on [81]. The p-value was calculated using the one-sample t-test in SPSS and calibrated using the Benjamini-Hochberg false discovery rate (FDR) correction [82]. The 95% confidence intervals (CI±; lower and upper bound) were calculated according to , where Ex is the efficiency of the reaction x. The μ, σ, CI and p-value for all qPCR experiments are shown in additional file 1. The mean μ of the normalized cycle difference was used to calculate the fold-change of expression using Ex (Additional file 4).

Phylogenetic analysis

RLK kinase domains were identified using PrositeScan http://au.expasy.org/tools/scanprosite/ webcite. Sequence alignments were performed using the ClustalW2 program [83]. Neighbour-joining trees were constructed with 1000 bootstrap sets using the Mega4 software package [84].

Micro-array analysis

Affymetrix raw data was downloaded from NASCArrays http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl webcite (accession number NASCARRAYS-143, paraquat; NASCARRAYS-353, ZAT12; NASCARRAYS-176, ABA time course experiment 1; NASCARRAYS-192, Ibuprofen), ArrayExpress http://www.ebi.ac.uk/microarray-as/ae/ webcite(accession numbers E-GEOD-12856, Blumeria graminis sp. hordei; E-GEOD-5684, Botrytis cinerea; E-ATMX-13, Methyl Jasmonate; E-MEXP-739, Syringolin A; E-MEXP-1797, Rotenone), Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/ webcite(accession numbers GSE5615, Elicitors LPS, HrpZ, Flg22 and NPP1; GSE5685, Virulent and avirulent Pseudomonas syringae:; GSE9955, BTH experiment 1, GDS417 E. cichoracearum; GSE5530, H2O2; GSE5722, O3; GSE12887, Norflurazon; GSE10732, OPDA and Phytoprostane; GSE7112, ABA experiment 2) and The Integrated Microarray Database System http://ausubellab.mgh.harvard.edu/imds webcite (Experiment name: BTH time course, BTH experiment 2).

The raw Affymetrix data was preprocessed with RMA using probe set annotations (custom cdf files) from http://brainarray.mbni.med.umich.edu/ webcite, version 11.0.1. Biological repeats of each experiment were combined by computing a mean of the measured gene expression. Gene expression was summarized by computing a log2 ratio of the treatment and control expressions (differential expression, DE). A visualization of the DE values is shown in Figure 4. Variation of differential expression in an experiment e, , was estimated by summing the variances of (logarithm of) treatment and control gene expressions.

Parametric bootstrapping was implemented by generating 1000 samples for each experiment and each gene from a Gaussian distribution with the estimated DE as the mean and as the variance.

Bootstrap samples were discretized to down regulated (log2 DE < -1), no regulation (-1 < log2 DE < 1), and up regulated (log2 DE > 1) genes. Bayesian agglomerative hierarchical clustering algorithm was then applied to the discretized bootstrap data. The Bayesian hierarchical clustering algorithm computes the best number of clusters by Bayesian hypothesis testing. For each pair of genes (and experiments, depending on the clustering direction), the number of times they were assigned to the same cluster was computed. These gene (or experiment) similarities were then used as distances for computing the hierarchical clustering (ward method) shown in Figure 4.

Promoter analysis

TAIR 9 version of promoter sequences of 500 bases and 1000 bases upstream of the Arabidopsis genes was downloaded from http://www.arabidopsis.org/ webcite. A list of verified Arabidopsis promoter elements was taken from http://arabidopsis.med.ohio-state.edu/AtcisDB/bindingsites.html webcite[54]. The set of CRKs was divided into three groups (all, ozone up-regulated and ozone down-regulated) and the plus and minus strands of the promoters were searched for significant enrichment of single promoter elements or a combination of two promoter elements in either of the strands. Fisher exact test with false discovery rate correction (q-values; [55]) was used for measuring the significance of the enrichment; q-value of 0.05 was used as the threshold.

Authors' contributions

MW, MB, SR, SK, BK and JK designed research. MW, MB, JS, NI, SLK and SM carried out research. MW, MB, JS and JK analyzed the data. MW, MB and JK wrote the paper. All authors have read and approved the final manuscript.

Acknowledgements

We would like to thank Dr. Sarah Coleman, Dr. Pinja Jaspers, Dr. Adrien Gauthier, Dr. Kirk Overmyer and Dr. Jorma Vahala for feedback, discussion and critical reading of the manuscript. Tuomas Puukko is thanked for excellent technical assistance. The work was supported by the Finnish Academy Centre of Excellence Programme (2006-2011). MW is supported by a Helsinki University Postdoctoral Researcher Grant. MB was supported by an Academy of Finland Post-doctoral grant (# 108760). SLK is supported by the Academy of Finland project # 130595. SK is financed from Welcome 2008/1 project operated within the Foundation for Polish Science Welcome Program co-financed by the European Regional Development Fund. SM is supported by the International Max-Planck-Research School (IMPRS). The project was carried out within the framework of the ERA-PG research program in the PROSIG consortium with funding from the Academy of Finland and the Deutsche Forschungsgemeinschaft.

References

  1. Shiu SH, Bleecker AB: Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis.

    Plant Physiol 2003, 132(2):530-43. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  2. Shiu SH, Karlowski WM, Pan R, Tzeng YH, Mayer KFX, Li WH: Comparative analysis of the receptor-like kinase family in Arabidopsis and rice.

    Plant Cell 2004, 16(5):1220-34. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  3. Shiu SH, Bleecker AB: Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases.

    Proc Natl Acad Sci USA 2001, 98(19):10763-8. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  4. Shiu SH, Bleecker AB: Plant receptor-like kinase gene family: diversity, function, and signaling.

    Sci STKE 2001, 2001(113):RE22. PubMed Abstract | Publisher Full Text OpenURL

  5. Ogawa M, Shinohara H, Sakagami Y, Matsubayashi Y: Arabidopsis CLV3 peptide directly binds CLV1 ectodomain.

    Science 2008, 319(5861):294. PubMed Abstract | Publisher Full Text OpenURL

  6. Escobar-Restrepo JM, Huck N, Kessler S, Gagliardini V, Gheyselinck J, Yang WC, Grossniklaus U: The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception.

    Science 2007, 317(5838):656-60. PubMed Abstract | Publisher Full Text OpenURL

  7. Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB: Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea.

    Proc Natl Acad Sci USA 1991, 88(19):8816-20. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  8. Stein JC, Dixit R, Nasrallah ME, Nasrallah JB: SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

    Plant Cell 1996, 8(3):429-45. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  9. van Zanten M, Snoek LB, Proveniers MCG, Peeters AJM: The many functions of ERECTA.

    Trends Plant Sci 2009, 14(4):214-8. PubMed Abstract | Publisher Full Text OpenURL

  10. Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC: BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.

    Cell 2002, 110(2):213-22. PubMed Abstract | Publisher Full Text OpenURL

  11. Nam KH, Li J: BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling.

    Cell 2002, 110(2):203-12. PubMed Abstract | Publisher Full Text OpenURL

  12. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nürnberger T, Jones JDG, Felix G, Boller T: A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence.

    Nature 2007, 448(7152):497-500. PubMed Abstract | Publisher Full Text OpenURL

  13. Heese A, Hann DR, Gimenez-Ibanez S, Jones AME, He K, Li J, Schroeder JI, Peck SC, Rathjen JP: The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants.

    Proc Natl Acad Sci USA 2007, 104(29):12217-22. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  14. Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G: Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

    Cell 2006, 125(4):749-60. PubMed Abstract | Publisher Full Text OpenURL

  15. Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH, Fauquet C, Ronald P: A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21.

    Science 1995, 270(5243):1804-6. PubMed Abstract | Publisher Full Text OpenURL

  16. Wang GL, Ruan DL, Song WY, Sideris S, Chen L, Pi LY, Zhang S, Zhang Z, Fauquet C, Gaut BS, Whalen MC, Ronald PC: Xa21D encodes a receptor-like molecule with a leucine-rich repeat domain that determines race-specific recognition and is subject to adaptive evolution.

    Plant Cell 1998, 10(5):765-79. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Xu WH, Wang YS, Liu GZ, Chen X, Tinjuangjun P, Pi LY, Song WY: The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase.

    Plant J 2006, 45(5):740-51. PubMed Abstract | Publisher Full Text OpenURL

  18. Lee SW, Han SW, Sririyanum M, Park CJ, Seo YS, Ronald PC: A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity.

    Science 2009, 326(5954):850-24. PubMed Abstract | Publisher Full Text OpenURL

  19. Chen K, Fan B, Du L, Chen Z: Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis.

    Plant Mol Biol 2004, 56(2):271-83. PubMed Abstract | Publisher Full Text OpenURL

  20. Czernic P, Visser B, Sun W, Savouré A, Deslandes L, Marco Y, Van Montagu M, Verbruggen N: Characterization of an Arabidopsis thaliana receptor-like protein kinase gene activated by oxidative stress and pathogen attack.

    Plant J 1999, 18(3):321-7. PubMed Abstract | Publisher Full Text OpenURL

  21. Chen Z: A superfamily of proteins with novel cysteine-rich repeats.

    Plant Physiol 2001, 126(2):473-6. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Chen K, Du L, Chen Z: Sensitation of defense responses and activation of programmed cell death by a pathogen-induced receptor-like protein kinase in Arabidopsis.

    Plant Mol Biol 2003, 53:61-74. PubMed Abstract | Publisher Full Text OpenURL

  23. Acharya BR, Raina S, Maqbool SB, Jagadeeswaran G, Mosher SL, Appel HM, Schultz JC, Klessig DF, Raina R: Overexpression of CRK13, an Arabidopsis cysteine-rich receptor-like kinase, results in enhanced resistance to Pseudomonas syringae.

    Plant J 2007, 50(3):488-99. PubMed Abstract | Publisher Full Text OpenURL

  24. Overmyer K, Brosché M, Kangasjärvi J: Reactive oxygen species and hormonal control of cell death.

    Trends Plant Sci 2003, 8(7):335-42. PubMed Abstract | Publisher Full Text OpenURL

  25. Apel K, Hirt H: Reactive oxygen species: metabolism, oxidative stress, and signal transduction.

    Annu Rev Plant Biol 2004, 55:373-99. PubMed Abstract | Publisher Full Text OpenURL

  26. Miller G, Shulaev V, Mittler R: Reactive oxygen signaling and abiotic stress.

    Physiol Plant 2008, 133(3):481-9. PubMed Abstract | Publisher Full Text OpenURL

  27. Mehler AH: Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents.

    Arch Biochem 1951, 33:65-77. PubMed Abstract | Publisher Full Text OpenURL

  28. Bartoli CG, Pastori GM, Foyer CH: Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.

    Plant Physiol 2000, 123:335-44. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  29. Bechtold U, Richard O, Zamboni A, Gapper C, Geisler M, Pogson B, Karpinski S, Mullineaux PM: Impact of chloroplastic- and extracellular-sourced ROS on high light-responsive gene expression in Arabidopsis.

    J Exp Bot 2008, 59(2):121-33. PubMed Abstract | Publisher Full Text OpenURL

  30. Gadjev I, Vanderauwera S, Gechev TS, Laloi C, Minkov IN, Shulaev V, Apel K, Inzé D, Mittler R, Van Breusegem F: Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis.

    Plant Physiol 2006, 141(2):436-45. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  31. Kim C, Meskauskiene R, Apel K, Laloi C: No single way to understand singlet oxygen signalling in plants.

    EMBO Rep 2008, 9(5):435-9. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  32. Joo JH, Wang S, Chen JG, Jones AM, Fedoroff NV: Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone.

    Plant Cell 2005, 17(3):957-70. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  33. Santner A, Estelle M: Recent advances and emerging trends in plant hormone signalling.

    Nature 2009, 459(7250):1071-8. PubMed Abstract | Publisher Full Text OpenURL

  34. Grant MR, Jones JDG: Hormone (dis)harmony moulds plant health and disease.

    Science 2009, 324(5928):750-52. PubMed Abstract | Publisher Full Text OpenURL

  35. Adie BAT, Pérez-Pérez J, Pérez-Pérez MM, Godoy M, Sánchez-Serrano JJ, Schmelz EA, Solano R: ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis.

    Plant Cell 2007, 19(5):1665-81. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  36. Fan J, Hill L, Crooks C, Doerner P, Lamb C: Abscisic acid has a key role in modulating diverse plant-pathogen interactions.

    Plant Physiol 2009, 150(4):1750-61. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  37. Ton J, Flors V, Mauch-Mani B: The multifaceted role of ABA in disease resistance.

    Trends Plant Sci 2009, 14(6):310-7. PubMed Abstract | Publisher Full Text OpenURL

  38. Galvez-Valdivieso G, Fryer MJ, Lawson T, Slattery K, Truman W, Smirnoff N, Asami T, Davies WJ, Jones AM, Baker NR, Mullineaux PM: The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.

    Plant Cell 2009, 21(7):2143-62. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  39. Overmyer K, Tuominen H, Kettunen R, Betz C, Langebartels C, Sandermann H, Kangasjärvi J: Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death.

    Plant Cell 2000, 12(10):1849-62. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  40. Lehti-Shiu MD, Zou C, Hanada K, Shiu SH: Evolutionary history and stress regulation of plant receptor-like kinase/pelle genes.

    Plant Physiol 2009, 150:12-26. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  41. Swarbreck D, Wilks C, Lamesch P, Beradrini TZ, Garcia-Hernandez M, Foerster H, Li D, Meyer T, Muller R, Ploetz L, Radenbaugh A, Singh S, Swing V, Tissier C, Zhang P, Huala E: The Arabidopsis Information Resource (TAIR): gene structure and function annotation.

    Nucleic Acids Res 2008, 36:D1009-D1014. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  42. Mühlenbock P, Szechynska-Hebda M, Plaszczyca M, Baudo M, Mateo A, Mullineaux PM, Parker JE, Karpinska B, Karpinski S: Chloroplast signaling and LESION SIMULATING DISEASE1 regulate crosstalk between light acclimation and immunity in Arabidopsis.

    Plant Cell 2008, 20(9):2339-56. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  43. Savage RS, Heller K, Xu Y, Ghahramani Z, Truman WM, Grant M, Denby KJ, Wild DL: R/BHC: fast Bayesian hierarchical clustering for microarray data.

    BMC Bioinformatics 2009, 10:242. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  44. Bhattacharjee A, Richards WG, Staunton J, Li C, Monti S, Vasa P, Ladd C, Beheshti J, Bueno R, Gillette M, Loda M, Weber G, Mark EJ, Lander ES, Wong W, Johnson BE, Golub TR, Sugarbaker DJ, Meyerson M: Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses.

    Proc Natl Acad Sci USA 2001, 98(24):13790-5. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  45. Clough SJ, Fengler KA, Yu IC, Lippok B, Smith RK, Bent AF: The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotide-gated ion channel.

    Proc Natl Acad Sci USA 2000, 97(16):9323-8. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  46. Jones JDG, Dangl JL: The plant immune system.

    Nature 2006, 444(7117):323-9. PubMed Abstract | Publisher Full Text OpenURL

  47. Yu IC, Parker J, Bent AF: Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant.

    Proc Natl Acad Sci USA 1998, 95(13):7819-24. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  48. Genger RK, Jurkowski GI, McDowell JM, Lu H, Jung HW, Greenberg JT, Bent AF: Signaling pathways that regulate the enhanced disease resistance of Arabidopsis "Defense, No Death" mutants.

    Plant Mol Biol 2008, 21(10):1285-96. OpenURL

  49. Penninckx IAMA, Thomma BPHJ, Buchala A, Métraux JP, Broekaert WF: Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis.

    Plant Cell 1998, 10(12):2103-113. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  50. Mang HG, Laluk KA, Parsons EP, Kosma DK, Cooper BR, Park HC, AbuQamar S, Boccongelli C, Miyazaki S, Consiglio F, Chilosi G, Bohnert HJ, Bressan RA, Mengiste T, Jenks MA: The Arabidopsis RESURRECTION1 gene regulates a novel antagonistic interaction in plant defense to biotrophs and necrotrophs.

    Plant Physiol 2009, 151:290-305. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  51. Ahlfors R, Brosché M, Kollist H, Kangasjärvi J: Nitric oxide modulates ozone-induced cell death hormone biosynthesis and gene expression in Arabidopsis thaliana.

    Plant J 2009, 58:1-12. Publisher Full Text OpenURL

  52. Sasaki-Sekimoto Y, Taki N, Obayashi T, Aono M, Matsumoto F, Sakurai N, Suzuki H, Hirai MY, Noji M, Saito K, Masuda T, Takamiya K, Shibata D, Ohta H: Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis.

    Plant J 2005, 44(4):653-68. PubMed Abstract | Publisher Full Text OpenURL

  53. Blanco F, Salinas P, Cecchini N, Jordana X, Hummelen PV, Alvarez ME, Holuigui L: Early genomic responses to salicylic acid in Arabidopsis.

    Plant Mol Biol 2009, 70(1-2):79-102. PubMed Abstract | Publisher Full Text OpenURL

  54. Palaniswamy SK, James S, Sun H, Lamb RS, Davuluri RV, Grotewold E: AGRIS and AtRegNet. a platform to link cis-regulatory elements and transcription factors into regulatory networks.

    Plant Physiol 2006, 140(3):818-29. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  55. Storey JD: A direct approach to false discovery rates.

    Journal of the Royal Statistical Society: Series B (Statistical Methodology) 2002, 64(3):479-98. Publisher Full Text OpenURL

  56. Dong J, Chen C, Chen Z: Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response.

    Plant Mol Biol 2003, 51:21-37. PubMed Abstract | Publisher Full Text OpenURL

  57. Gómez-Gómez L, Boller T: FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis.

    Mol Cell 2000, 5(6):1003-11. PubMed Abstract | Publisher Full Text OpenURL

  58. Sun X, Cao Y, Yang Z, Xu C, Li X, Wang S, Zhang Q: Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein.

    Plant J 2004, 37(4):517-27. PubMed Abstract | Publisher Full Text OpenURL

  59. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T: Bacterial disease resistance in Arabidopsis through flagellin perception.

    Nature 2004, 428(6984):764-7. PubMed Abstract | Publisher Full Text OpenURL

  60. Sivaguru M, Ezaki B, He ZH, Tong H, Osawa H, Baluska F, Volkmann D, Matsumoto H: Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis.

    Plant Physiol 2003, 132(4):2256-66. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  61. Osakabe Y, Maruyama K, Seki M, Satou M, Shinozaki K, Yamaguchi-Shinozaki K: Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis.

    Plant Cell 2005, 17(4):1105-19. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  62. Chae L, Sudat S, Dudoit S, Zhu T, Luan S: Diverse transcriptional programs associated with environmental stress and hormones in the Arabidopsis Receptor-Like Kinase gene family.

    Mol Plant 2009, 2:84-107. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  63. Grant JJ, Loake GJ: Role of reactive oxygen intermediates and cognate redox signaling in disease resistance.

    Plant Physiol 2000, 124:21-9. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  64. Langebartels C, Ernst D, Kangasjärvi J, Sandermann H: Ozone effects on plant defense.

    Methods Enzymol 2000, 319:520-35. PubMed Abstract | Publisher Full Text OpenURL

  65. Ivanov B, Asada K, Edwards GE: Analysis of donors of electrons to photosystem I and cyclic electron flow be redox kinetics of P700 in chloroplasts isolated bundle sheath strands of maize.

    Photosynth Res 2007, 92:65-74. PubMed Abstract | Publisher Full Text OpenURL

  66. Tamaoki M, Nakajima N, Kubo A, Aono M, Matsuyama T, Saji H: Transcriptome analysis of O3-exposed Arabidopsis reveals that multiple signal pathways act mutually antagonistically to induce gene expression.

    Plant Mol Biol 2003, 53(4):443-56. PubMed Abstract | Publisher Full Text OpenURL

  67. Ali R, Ma W, Lemtiri-Chlieh F, Tsaltas D, Leng Q, von Bodman S, Berkowitz GA: Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity.

    Plant Cell 2007, 19(3):1081-95. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  68. Evans NH, McAinsh MR, Hetherington AM, Knight MR: ROS perception in Arabidopsis thaliana: the ozone-induced calcium response.

    Plant J 2005, 41(4):615-26. PubMed Abstract | Publisher Full Text OpenURL

  69. Ma W, Smiegel A, Verma R, Berkowitz GA: Cyclic nucleotidegated channels and related signaling components in plant innate immunity.

    Plant Signal Behav 2009, 4(4):272-82. Publisher Full Text OpenURL

  70. Overmyer K, Brosché M, Pellinen R, Kuittinen T, Tuominen H, Ahlfors R, Keinänen M, Saarma M, Scheel D, Kangasjärvi J: Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant.

    Plant Physiol 2005, 137(3):1092-104. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  71. Carrera J, Rodrigo G, Jaramillo A, Elena S: Reverse-engineering Arabidopsis thaliana transcriptional network under changing environmental conditions.

    Genome Biol 2009, 10(9):R96. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  72. Zipfel C: Early molecular events in PAMP-triggered immunity.

    Curr Opin Plant Biol 2009, 12(4):414-20. PubMed Abstract | Publisher Full Text OpenURL

  73. Chen H, Xue L, Chintamanani S, Germain H, Lin H, Cui H, Cai R, Zuo J, Tang X, Li X, Guo H, Zhou JM: ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 repress SALICYLIC ACID INDUCTION DEFICIENT2 expression to negatively regulate plant innate immunity in Arabidopsis.

    Plant Cell 2009, 21(8):2527-40. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  74. Liu Y, Ren D, Pike S, Pallardy S, Gassmann W, Zhang S: Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade.

    Plant J 2007, 51(6):941-54. PubMed Abstract | Publisher Full Text OpenURL

  75. Zurbriggen MD, Carrillo N, Tognetti VB, Melzer M, Peisker M, Hause B, Hajirezaei MR: Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-host interaction between tobacco and Xanthomonas campestris pv. vesicatoria.

    Plant J 2009, 60:962-73. PubMed Abstract | Publisher Full Text OpenURL

  76. Miller G, Schlauch K, Tam R, Cortes D, Torres MA, Shulaev V, Dangl JL, Mittler R: The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli.

    Sci Signal 2009, 2(84):ra45. PubMed Abstract | Publisher Full Text OpenURL

  77. Vahisalu T, Puzõrjova I, Brosché M, Valk E, Lepiku M, Moldau H, Pechter P, Wang YS, Lindgreen O, Salojärvi J, Loog M, Kangasjärvi J, Kollist H: Ozone-triggered rapid stomatal response involves production of reactive oxygen species and is controlled by SLAC1 and OST1.

    Plant J 2010, 62:442-453.

    Accepted article doi: 10.1111/j.1365-313X.2010.04159.x

    PubMed Abstract | Publisher Full Text OpenURL

  78. Mateo A, Mühlenbock P, Rustérucci C, Chang CCC, Miszalski Z, Karpinska B, Parker JE, Mullineaux PM, Karpinski S: LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy.

    Plant Physiol 2004, 136:2818-30. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  79. Wrzaczek M, Brosché M, Kollist H, Kangasjärvi J: Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

    Proc Natl Acad Sci USA 2009, 106(13):5412-7. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  80. Vandesompele J, Preter KD, Pattyn F, Poppe B, Roy NV, Paepe AD, Spelemann F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.

    Genome Biol 2002, 3(7):0034.1-0034.11. BioMed Central Full Text OpenURL

  81. Applied-Biosystems: Relative quantitation of gene expression: ABI PRISM 7700 Sequence Detection System. User bulletin #2: Rev. B.

    Weiterstadt, Germany, Applied Biosystems 2001. OpenURL

  82. Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing.

    J Royal Stat Soc, Series B 1995, 57:289-300. OpenURL

  83. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0.

    Bioinformatics 2007, 23(21):2947-8. PubMed Abstract | Publisher Full Text OpenURL

  84. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.

    Mol Biol Evol 2007, 24(8):1596-9. PubMed Abstract | Publisher Full Text OpenURL