JXB Advance Access originally published online on September 22, 2009
Journal of Experimental Botany 2009 60(15):4383-4396; doi:10.1093/jxb/erp277
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© 2009 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/2.5/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 |
Arabidopsis L-type lectin receptor kinases: phylogeny, classification, and expression profiles
Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen and Graduate School Experimental Plant Sciences, The Netherlands
* To whom correspondence should be addressed: E-mail: francine.govers{at}wur.nl
Received 14 July 2009; Revised 8 August 2009 Accepted 17 August 2009
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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In plants, lectin receptor kinases are considered to play crucial roles during development and in the adaptive response to various stimuli. Arabidopsis lectin receptor kinases can be divided into three type-classes based on sequence similarity of their extracellular lectin motifs. The current study focuses on the legume-like lectin receptor kinases (LecRKs), which are regarded as ideal candidates for monitoring cell wall integrity and are possibly functional in adaptive responses. An inventory of the Arabidopsis LecRK gene family is presented here. It consists of 45 members including three that were recently identified; two encode N-terminal truncated variants one of which has two in tandem kinase domains. Phylogenetic trees derived from full-length amino acid sequence alignments were highly concordant to phylograms that were purely based on lectin motifs or kinase domains. The phylograms allowed reclassification of the LecRK genes and hence a new proposal for gene nomenclature was suggested. In addition, a comprehensive expression analysis was executed by exploring public repositories. This revealed that several LecRK genes are differentially expressed during plant growth and development. Moreover, multiple LecRKs appear to be induced upon treatment with elicitors and pathogen infection. Variation in gene expression was also analysed in seedlings of diverse Arabidopsis accessions. Taken together, this study provides a genome-wide overview of the LecRK gene family and an up-to-date classification using a novel and systematic gene nomenclature.
Key words: Arabidopsis, evolutionary relationship, LecRKs, nomenclature, RLK
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Plants are facing diverse developmental and environmental stimuli. Perception and transduction of these signals is largely governed by so-called receptor-like kinases (RLKs). The Arabidopsis genome harbours over 600 RLK genes, many of which can be grouped into distinct subfamilies based on their extracellular domains (Shiu and Bleecker, 2001a, 2003). The variability in the extracellular domain organization reflects their diverse function and mode of signal perception. For most RLKs, however, neither their ligands nor their downstream targets are known. Examples of elucidated ligand–receptor pairs include the pattern recognition receptors FLS2 and EFR that interact with the microbial associated molecular patterns (MAMPs) flg22 and EF-Tu, respectively, as well as the plant development-associated receptors CLV1, which has the polypeptide CLV3 as ligand, and BRI1 that functions in brassinosteroid perception (Gómez-Gómez and Boller, 2000; Kinoshita et al., 2005; Zipfel et al., 2006; Ogawa et al., 2008).
The adaptive responses of plants to extracellular ligands and stimuli are governed by a functional continuum between the plant cell wall (CW) and the plasma membrane (PM) in which RLKs with CW-bound extracellular domains probably function as cell wall integrity sensors (reviewed by Humphrey et al., 2007). The Arabidopsis wall-associated kinases (WAKs) are thought to operate as physical CW-PM linkers (Anderson et al., 2001; Verica and He, 2002). By means of various biochemical techniques it was shown that extracellular subdomains of WAK1 interact with a secreted glycine-rich protein (AtGRP3) and with cell wall pectins (Park et al., 2001; Decreux and Messiaen, 2005). Arabidopsis plants with impaired WAK4 expression were found to be affected in cell elongation and morphology (Lally et al., 2001; Kohorn et al., 2006a). Moreover, inactivation of WAK2, one of the Arabidopsis WAK genes, reduced the activity of vacuolar invertase that functions in controlling cell turgor, and hence seems to regulate cell expansion (Kohorn et al., 2006b). Other Arabidopsis RLKs that may have a role in binding cell wall components are the PERKs and members of the CrRLK1L family closely resembling a RLK from Catharanthus roseus (Madagascar periwinkle) (Schulze-Muth et al., 1996). PERKs harbour extracellular proline-rich sequences that share similarity with plant extensins, suggesting a direct interaction with cell wall components (Nakhamchik et al., 2004), whereas THESEUS1 – a CrRLK1L protein from Arabidopsis – is activated in mutants deficient in cellulose and triggers growth inhibition and ectopic lignin accumulation (Hématy et al., 2007).
Another group of RLKs that are regarded as potential CW-PM linkers are the lectin receptor kinases which contain extracellular lectin motifs that are known to bind various carbohydrates. Based on the extracellular lectin motifs three types can be distinguished; G, C, and L (Fig. 1). Arabidopsis has around 40 RLK genes encoding bulb-associated lectin receptor kinases. Previously these were categorized as B-type lectin receptor kinases because their lectin motif resembles mannose-specific lectins of bulb species. Because these lectins are no longer confined to bulbs Van Damme et al. (2007) proposed to rename them as GNA-related lectins, here abbreviated as G-type lectins. Well-studied G-type lectin receptor kinases are the S-locus receptor kinases, historically known as S-domain RLKs after the functionality of the ectodomain in self-incompatibility (SI) in flowering plants. In Brassicaceae, the S-locus receptor kinase SRK acts as the stigmatic determinant of the SI response and possibly functions as a receptor for the pollen ligand SCR/SP11. The shift to self-fertility in Arabidopsis thaliana is partly due to non-functional SRK sequences (Kusaba et al., 2001; Sherman-Broyles et al., 2007). G-type lectin receptor kinases were also shown to play roles in plant defence. The rice gene Pi-d2, for example, confers resistance to the fungal pathogen Magnaporthe grisea whereas NgRLK1 from Nicotiana glutinosa was selected in a yeast two-hybrid screen as a putative interactor with capsicein, an elicitin from Phytophthora capsici (Chen et al., 2006; Kim et al., 2009). Besides the G-type lectin motifs the extracellular domains of these proteins contain cysteine-rich EGF-like (epidermal growth factor) and PAN (plasminogen-apple-nematode) motifs that both function in protein homodimerization (Naithani et al., 2007). As yet, the role of the G-lectin motif is unknown and there is also no evidence for a function in ligand binding.
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C-type (calcium-dependent) lectin motifs can be found in a large number of mammalian proteins that mediate innate immune responses such as antigen uptake and T-cell interaction and play a major role in pathogen recognition (reviewed by Cambi et al., 2005). Unlike the omnipresent nature of C-type lectins in mammals, they seem to be scarce in plants. Arabidopsis has only a single gene encoding a protein with a C-type lectin motif but so far its function has not been elucidated.
The third type comprises the legume-like or L-type lectin receptor kinases (LecRKs). Their extracellular domains resemble soluble legume lectins which are ubiquitous in leguminous seeds. For several legume lectins the 3D-structure and their carbohydrate-protein binding specificity has been elucidated. The sequence similarity to legume lectins prompted the hypothesis that the potential ligands of LecRKs are oligosaccharides (André et al., 2005). Molecular modelling showed that the sugar-binding residues in LecRKs are poorly conserved and it is therefore unlikely that these receptors bind monosaccharide molecules. There is, however, a rather conserved hydrophobic-binding site, and hence LecRKs may serve in the recognition of small hydrophobic ligands, such as plant hormones or MAMPs (Barre et al., 2002; André et al., 2005).
Although LecRKs are implied to function in diverse biological processes, their exact biological role has not yet been clarified. LecRKs that have been described to function during plant development include the SGC lectin RLK of Arabidopsis, which is required for proper pollen development (Wan et al., 2008) and MtLecRK1;1 from the nitrogen-fixing legume Medicago truncatula that influences nodulation (Navarro-Gochicoa et al., 2003). Expression of the Arabidopsis lectin receptor kinase lecRK-a1 was shown to be induced during wounding, senescence of leaves, and in response to oligogalacturonides, which might be released upon disruption of the plant cell wall (Riou et al., 2002). More recently, Xin et al. (2009) showed that a specific subfamily of LecRKs is responsible for negatively regulating the abscisic acid (ABA) response during seed germination and hypothesized that these genes directly or indirectly affect defence. Lately, several reports have presented data linking ABA-signalling with defence responses (Asselbergh et al., 2008). NbLRK1, a legume-like lectin receptor kinase from Nicotiana benthamiana was reported to interact intracellularly with the P. infestans elicitin INF1 and seems to be involved in the subsequent INF1-induced cell death (Kanzaki et al., 2008). Another LecRK with a potential link to plant defence and pathogen response is LecRK79 in Arabidopsis. It interacts via its lectin motif with the tripeptide motif RGD (arginine-glycine-aspartic acid) in IPI-O, an RXLR effector from the late blight pathogen Phytophthora infestans that can disrupt CW-PM adhesions through the RGD motif (Senchou et al., 2004; Gouget et al., 2006). LecRK79 mediates CW-PM adhesions and hence the continuum between the cell wall and the plasma membrane (Gouget et al., 2006). Its role in plant defence is, furthermore, supported by the observation that LecRK79 expression is induced upon inoculation with several non-host and avirulent pathogens (Bouwmeester et al., 2008). Taken together, these data suggests that LecRKs play crucial roles in both developmental and adaptive processes.
Over the past years several Arabidopsis LecRKs have been investigated. Gene naming, however, has not been uniform and this has sometimes resulted in confusion and miscommunication. Some LecRK genes have multiple names, whereas the majority has only an AGI gene code. Gene names are often based on protein functionality or molecular mass, but this does not reflect their phylogenetic relationship. Unfortunately, several gene names have been chosen based on incomplete and not always informative gene classification. For example, LecRK-a4 – named by Hervé et al. (1996) – strongly resembles LecRKA4.1, the gene name recently assigned by Xin et al. (2009) to a LecRK that regulates the ABA response.
In this study, the information available on all members of the Arabidopsis L-type lectin receptor kinase gene family has been collated and summarized and a bioinformatic analysis has been performed to clarify their relationship. Based on the revised phylogenetic classification, a simplified nomenclature for the Arabidopsis LecRKs is proposed, which could serve as a basis for gene naming in other plant species and will hopefully improve communication among scientists working on this group of plant receptor kinases. A comprehensive survey of publicly available expression data has also been undertaken. The expression profiles show that LecRKs are activated by various biotic and abiotic stimuli and are differentially expressed in various Arabidopsis accessions.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Sequence database search and gene analysis
Gene sequences were retrieved using the available servers at the TAIR website (http://www.arabidopsis.org). Additional BLAST searches were conducted on the Arabidopsis genome sequence and proteome at TAIR and MIPS (http://mips.gsf.de). All genes were checked for annotation mistakes. Structural domain prediction was performed using the SMART (http://smart.embl-heidelberg.de) and Pfam (http://pfam.sanger.ac.uk) databases.
SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) was used for the prediction of signal peptides. The web servers TMHMM (http://www.cbs.dtu.dk/services/TMHMM), SOSUI (http://bp.nuap.nagoya-u.ac.jp/
sosui/), and PRED-TMR2 (http://athina.biol.uoa.gr/PRED-TMR2/) were used to predict transmembrane regions. Additional domain prediction and verification was performed using the programs InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/) and ProDom (http://prodom.prabi.fr). Homology at protein and nucleotide levels was determined by pairwise sequence comparison with the AlignX module in Vector NTI® (Invitrogen).
Alignment and phylogenetic analysis of LecRK genes
Full-length protein and domain sequences, as defined in the previous section, were compiled and aligned using ClustalW (Larkin et al., 2007). GONNET with a penalty gap opening of 10 and a gap extension of 0.1 was used as the protein weight matrix. The obtained sequence alignments were used as input to construct phylogenetic trees with the minimal evolution algorithm within MEGA 4 (Tamura et al., 2007). Poisson correction was used to account for multiple substitutions and alignment gaps were removed in a pairwise fashion. The statistical strength was assessed by bootstrap resampling using 10 000 replicates.
Analyses conducted with different algorithms within MEGA 4 generated comparable phylograms. Protein phylogenetic trees were rooted with amino acid sequences of the Arabidopsis RLKs WAK1 (At1g21250), PERK1 (At3g24550), the G-type lectin receptor kinases (SRKs) ARK1 (At1g65790) and CES101 (At3g16030), and the C-type lectin receptor kinase encoded by the single-copy gene At1g52310. Domain-based phylogenetic trees were rooted with the predicted extra- and intracellular domains of these outgroup genes. Branches corresponding to partitions reproduced in <50% bootstrap replicates were collapsed.
Analysis of expression data
Arabidopsis EST and cDNA data was retrieved from the TAIR website. MPSS signature data were retrieved from the MPSS database (http://mpss.udel.edu/at/) (Lu et al., 2005). Sums of abundance of sense strand signatures (classes 1, 2, 5, and 7) were regarded as significant and reliable MPSS expression data. Sequence queries in the 17 bp and 20 bp signature databases showed comparable expression profiles. Expression data was further analysed by using the Genevestigator V3 web tool (http://www.genevestigator.com) (Hruz et al., 2008). Expression analysis was restricted to gene chip experiments based on the sequenced genome of the Arabidopsis accession Col-0. LecRK-V.7 (At3g58740) and LecRK-V.8 (At3g59750) share the same probe-set (25148_s_at) and represent equal values. The blue colour codes displayed in Fig. 3 are based on the concept of expression potential, in which the darkest blue colour is representing the maximum expression level that a given gene can reach throughout all arrays available in the database. Because equal blue colours can depict different expression values, comparison of expression levels is only justified between one gene in different tissues and not between individual genes.
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Natural variation in LecRK expression between Arabidopsis accessions was determined by using the Arabidopsis eFP browser of the Bio-Array Resource (BAR) website (http://bar.utoronto.ca/) (Winter et al., 2007). Log2 expression values relative to the control value (Col-0) were tabulated and reformatted to a colour-scale with the BAR HeatMapper Plus Tool to visualize expression differences easily. Expression was measured in triplicate samples of aerial parts of 4-d-old seedlings of nine geographically separated Arabidopsis accessions; including the reference accession Col-0 (Columbia, USA), Van-0 (Vancouver, USA), Kin-0 (Kindalville, USA), Est-0 (Estonia), Bay-0, (Bayreuth, Germany), Nd-0 (Niederzenz, Germany), Ler-2 (Landsberg, Germany), Sha (Shahdara, Tajikistan), and Cvi (Cape Verde Islands).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Gene identification and protein structure prediction
L-type lectin receptor kinases (LecRKs) were identified by BLAST searches and by exploring previously constructed gene lists (Shiu and Bleecker, 2001b, 2003; Barre et al., 2002). In total, 45 LecRKs were identified, 42 of which were previously described by Barre et al. (2002) (Table 1). The three added in this survey are At5g60310, At3g46760, and At2g32800, which were previously listed as putative lectin receptor kinases in the Arabidopsis RLK inventory by Shiu and Bleecker (2001b, 2003). At5g60310 belongs to a cluster of three closely linked LecRK genes, and seems to encode a typical L-type lectin receptor kinase (Fig. 1). Protein sequence comparison revealed, however, that At5g60310 encodes an abnormal protein kinase lacking sub-domains X and XI, thus suggesting impaired kinase activity. The other two, At3g46760 and At2g32800, encode proteins with kinase domains but lacking transmembrane motifs and extracellular domains. Moreover, the kinase encoded by At3g46760 contains a shortened activation loop, which could affect proper kinase functioning. Structure prediction of At2g32800 revealed a peculiar arrangement of two repeated kinase domains (here named AT3G32800A and AT3G32800B) that contain all structural sub-domains. Proteins with two in-tandem kinase domains do exist in vertebrates and are known as Janus kinases (JAKs) based on their two-faced function: the first domain exhibits kinase activity while the other down-regulates this activity. JAKs appear to play a critical role in diverse processes such as cytokinin signalling in mammalian cells (reviewed by Yamaoka et al., 2004).
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Prediction analyses showed that most LecRKs have all the features of typical L-type lectin receptors, i.e. a putative signal sequence, an extracellular lectin motif, a transmembrane region, and a protein kinase domain (Fig. 1). In addition to the three exceptions described above, four other LecRKs encode structurally different proteins. The one encoded by At4g28350 was predicted to lack a transmembrane region, and those encoded by At2g29220, At2g29250, and At3g45390 seem to be devoid of a typical kinase structure (Table 1).
LecRK clusters and clades
To categorize the Arabidopsis LecRK genes, the LecRK gene family was divided into clusters and clades. A gene cluster represents physical proximity on the chromosome and was defined as two or more homologous genes within a maximum of eight adjacent gene models between individual members. Nine distinct LecRK gene clusters were found with a maximum of six genes per cluster (i.e. cluster 3A) (Table 1). The gene clusters are dispersed across the five Arabidopsis chromosomes, but the larger gene clusters are predominantly located on chromosome 3 and 5.
A gene clade was defined as a group of at least two homologous genes with a minimum of 50% identity at both the nucleotide and amino acid level (Mondragon-Palomino and Gaut, 2005). By means of pairwise comparison nine major LecRK clades were determined. The two largest LecRK clades comprise 11 and nine genes, respectively. Two clades contain four genes, and the five smallest ones consist of two LecRK genes. Seven LecRK genes do not belong to any clade and were assigned as singletons. The 28 LecRK genes distributed over clusters all belong to a clade. Hence, physically linked LecRK genes also contain a high pairwise identity (>50%). This is probably due to ancient duplication events and it is therefore not surprising that genes in one cluster belong to a single clade (Table 1).
LecRK clade phylogeny
To determine the evolutionary relationship between members of the LecRK protein family the protein sequences were aligned with ClustalW using GONNET as the protein weight matrix. Subsequently, phylograms were constructed with MEGA 4 using several algorithms, i.e. Neighbor–Joining, maximum parsimony, and minimal evolution. The robustness of the phylograms was determined by bootstrap analysis with 10 000 replications. The consensus topologies generated by the different algorithms were comparable (data not shown). All phylograms were rooted using the Arabidopsis RLKs WAK1, PERK1, the Arabidopsis C-type lectin receptor kinase (At1g52310), and the G-type lectin receptor kinases ARK1 and CES101 as outgroup representatives and were largely supported by high bootstrap values (Fig. 2).
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Phylogenetic analysis conducted with 43 out of 45 full-length LecRK proteins resulted in 38 LecRKs in nine distinct clades and five singletons, which is in line with the pairwise comparisons (Fig. 2A). In this analysis At2g32800 and At3g46760 were excluded because they lack extracellular domains. The phylogram based on the lectin domain shows comparable gene relationships and also similar grouping as depicted in the phylogenetic tree generated with the full-length protein sequences (Fig. 2B). Similarly, the phylogram based on the kinase domain is comparable. As expected the three kinase domains added in the phylogram At2g32800A, At2g32800B, and At3g46760, group with the LecRKs and not with the outgroups. Moreover, they do not group with any clade and even the two kinase domains within At2g32800 behave as singletons (Fig. 2C). Overall these phylograms suggest that the LecRK members of the same clade are highly identical to each other in both extracellular and intracellular domains. This feature can be used for the phylogenetic characterization of full-length and truncated LecRK protein sequences of other plant species.
Uniform nomenclature for LecRK gene members
The proposed gene nomenclature is based on the grouping of closely related LecRK genes within the evolutionary divergent clades. Clades were designated by the Roman numerals I to IX starting clockwise within the phylogram shown in Fig. 2A. The individual gene members within the clades were given Arabic numerals based on their physical map position. Singletons were also ordered based on their physical map position and numbered S.1 to S.7. Table 1 lists the revised and more consistent gene nomenclature. For comparison, the previous gene classification (Barre et al., 2002) and the alternative LecRK gene names are included.
LecRK genes are differentially expressed in various tissues and developmental stages
To obtain more insight into the expression patterns of the individual LecRKs during plant development, a comprehensive expression analysis was performed by making use of various on-line repositories. Based on the number of full-length cDNAs and ESTs retrieved from the TAIR website, it is apparent that the LecRK genes have variable expression profiles. For about one-third of the LecRK genes there are no ESTs, for another one-third there are fewer than 10 ESTs, whereas five of them have over 20 ESTs and thus seem to be highly expressed (Table 2). Transcript abundance was verified by analysing expression data generated by massively parallel signature sequencing (MPSS) (Lu et al., 2005). This showed that several gene members that lack or have few ESTs do have matching expression signatures (Table 2; see Supplementary Table S1 at JXB online). LecRK-V.3, for example, has no ESTs and low MPSS values in several Arabidopsis tissues, but displays a specific high MPSS value in the sup/ap1 immature inflorescence stage. Similarly, LecRK-V.6 lacks ESTs but MPSS signatures revealed a subtle expression in root tissue. Gene expression profiles based on microarray data (Fig. 3) also showed some inconsistencies with EST counts or MPSS signatures. Taken together, however, it can be concluded that several LecRK genes are not or hardly expressed in any of the Arabidopsis tissues or developmental stages, for example, several members of the LecRK-V clade and both LecRK-III genes (Fig. 3), whereas a few others are broadly expressed in all tissues, in particular LecRK-I.9, LecRK-IV.1, and LecRK-VIII.1.
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LecRK gene expression in response to various stimuli
Microarray data were also queried to reveal changes in LecRK expression upon treatment with various hormones, environmental stresses, elicitors and pathogens. As shown in Fig. 4 several LecRK genes, including some that are not or hardly expressed during plant development, were activated or repressed in response to diverse stimuli.
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Treatment of Arabidopsis with the hormones auxin and ethylene did not result in significant changes in expression (Fig. 4). By contrast, clear changes in transcript levels of some LecRK genes were detected in plants treated with ABA, brassinolide, cytokinin, methyl jasmonate, and salicylic acid (SA). MPSS signature data suggest an extremely high activity of LecRK-IV.1 at 52 h after SA application (Table 2; see Supplementary Table S1 at JXB online). The same, but to a lesser extent, is true for LecRK-VI.2. Both LecRK-IV.1 and LecRK-VI.2 showed differential expression in different (untreated) tissues (Fig. 3) but it seems that this is not correlated to SA sensitivity; other LecRK genes with differential expression during development were not activated by SA.
In addition, several abiotic stresses influenced LecRK expression (Fig. 4). In response to wounding, potassium deprivation, and salt stress there were hardly any changes. Osmotic stress, anoxia, and cold, resulted in minor overall changes with the exception of one or two genes that strongly responded to one particular treatment. An example is LecRK-V.6 which was strongly repressed by cold treatment. In the case of drought many of the LecRK genes responded, two of which were highly stimulated and two others strongly repressed. Response to treatment with hydrogen peroxide was particularly associated with changes in expression levels of genes in clade VII, VIII, and IX and the same genes, as well as some additional ones, were induced by ozone treatment. The most severe changes were observed in the dark when about half of the LecRK genes was activated and showed much higher expression levels than the controls. In the presence of high CO2 concentrations the reverse was observed, with many of the LecRK genes showing a lower expression than the controls.
Upon treatment with a variety of elicitors, rather similar patterns were obtained (Fig. 4). Expression of several LecRK genes from different clades was strongly induced upon treatment with six out of seven elicitors. The exception is bacterial lipopolysaccharide (LPS) which hardly induced any LecRK apart from LecRK-IX.2. The genes that were most strongly induced are LecRK-I.8, LecRK-IV.2, LecRK-IV.3, LecRK-V.2, LecRK-V.5, LecRK-VI.2, LecRK-VI.3, LecRK-VII.2, and LecRK-IX.2. LecRK-VI.4 behaved remarkably; a strong repression by syringolin but a strong induction by five other elicitors.
Unlike the rather uniform expression pattern that is observed upon elicitor treatment, the patterns observed during interaction with biotic agents are much more diverse. The most strongest repression was measured upon interaction with the aphid Myzus persicae with 17 LecRK genes strongly down-regulated and none up-regulated. Interaction with another insect, the sweet potato whitefly Bemisia tabaci, only induced the activity of a few LecRK genes, and resulted in most cases in an increase in expression rather than a decrease. Virus infection influenced the expression of several LecRK genes, both positive and negative, and the patterns were specific for each of the two plant–virus interactions included in this analysis. No significant changes in gene expression were observed during interaction with the arbuscular mycorrhizal fungus Gigaspora rosea or the nematode Meloidogyne incognita. In addition, a compatible interaction with the powdery mildews Erysiphe cichoracearum or E. orontii did not alter LecRK expression substantially. By contrast, in an incompatible interaction with the non-host powdery mildew Blumeria graminis f. sp. hordei (synonym Erysiphe graminis) expression of several LecRK genes was induced or repressed. Similarly, expression was largely unaffected upon infection with the pathogenic bacterium Pseudomonas syringae DC3000, or its non-pathogenic type III secretion-defective mutant hrpA. However, in incompatible interactions resulting from infection with Pseudomonas strains secreting the effectors AvrRpt2, AvrRpm1 or AvrRps4, expression of several LecRK genes was found to be induced. Four of these, LecRK-I.8, LecRK-I.9, LecRK-V.5, and LecRK-IX.2, were also induced upon inoculation of Arabidopsis with the oomycete Phytophthora infestans. Arabidopsis is a non-host for P. infestans and this may explain why these particular LecRK genes were also strongly induced upon elicitor treatment. Inoculation of Arabidopsis with the grey mould fungus Botrytis cinerea results in necrotic lesions and, as observed in Fig. 4, this was accompanied by the induction or repression of a few particular LecRK genes including some that are affected by other pathogens.
LecRK genes are differentially expressed among Arabidopsis accessions
To examine whether LecRK genes are differentially expressed among Arabidopsis accessions, expression data were retrieved from the Bio-Array Resource (BAR) website using the Arabidopsis Natural Variation eFP Browser. Expression values relative to Col-0 were retrieved for each LecRK gene member. Only microarray data generated from experiments that made use of triplicate sampling of plant material (aerial parts of 4-d-old seedlings) were analysed and this restricted the analysis to the Arabidopsis accessions Col-0, Van-0, Kin-0, Est-0, Bay-0, Nd-0, Ler-2, Sha, and Cvi. Transcript profiles were visualized with the BAR HeatMapper Plus. For most LecRK genes relatively similar expression levels were found among the various accessions (Fig. 5). Exceptions are LecRK-II.2, LecRK-V.5, LecRK-IV.7, and LecRK-IV.8, which seem to have a higher relative expression in all accessions when compared to Col-0. It should be noted that LecRK-IV.7 and LecRK-IV.8 hybridize to the same probe set resulting in equal signal intensities. The few genes with a low relative expression among the accessions are mainly in clade LecRK-VIII and LecRK-IX. Only a few LecRK genes were found to have a high expression value in one accession but a low expression in another accession. The most striking differences in expression values were found for LecRK-I.5 and LecRK-IV.3 both of which have substantially lower relative transcript levels in Ler-2 and LecRK-V.5 that has a higher expression in the accession Bay-0.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Several membrane-bound receptors in plants are shown to be functional in the perception of external signals and the subsequent triggering of downstream signal cascades to initiate adaptive responses. In this study, the focus was on the LecRK subfamily of membrane-bound receptors in Arabidopsis. The available gene information and expression data of the 45 members in this subfamily have been summarized and a new systematic nomenclature is proposed for the LecRK genes based on clades within the subfamily. Previously, Barre and co-workers (2002) conducted a similar analysis for 42 Arabidopsis LecRK genes, including structural alignments and molecular modelling of the encoded proteins. In that study, however, three genes that were predicted to encode LecRKs by Shiu and Bleeker (2001b, 2003) were not included. Here, the phylogenetic relationship within the LecRK gene family has been revised by constructing phylograms with more sophisticated statistical and computational methods. Three phylograms which were rooted with five outgroup RLKs and based on multi-alignments of the full-length proteins, lectin motifs, and kinase domains, respectively, showed clustering in nine different clades and seven singletons. Pairwise comparisons also resulted in the same nine clades and seven singletons and this consensus strengthens our clade division. The subdivision into clades and singletons formed the basis for the new, more systematic nomenclature and provides a helpful standard for gene identification and naming in other plant species.
The currently available expression datasets provide a basis to generate biological hypotheses as specific patterns of gene expression may give hints about their functionality. EST, MPSS, and microarray data indicate that the LecRK genes have variable expression patterns in different Arabidopsis tissues, developmental stages, in response to stimuli, and in various accessions. Several of the LecRKs have low transcript levels during plant development, but are induced by hormone or elicitor treatment or during interaction with pathogens. For example, a striking difference was found for the two genes in the LecRK-III clade. They are not expressed during plant development, but are expressed in response to biotic and abiotic stimuli. They do respond to different stimuli, though and, thus, their expression pattern is not clade specific. Overall, there was no correlation between the expression pattern of a particular gene and its position in the phylogenetic tree. For all 45 LecRK genes, including the ones encoding unusual protein structures, expression was observed in at least one of the conditions that was analysed, hence, demonstrating that none of them represents a non-functional dead copy. So far, functional analysis of the Arabidopsis LecRK genes based on knock-out mutants has been limited to 5 out of the 45 family members, i.e. LecRK-I.9, LecRK-IV.2, and three members of the LecRK-VI clade (K Bouwmeester et al., unpublished data; Wan et al., 2008; Xin et al., 2009). The phylogenetic relationship and the expression analysis presented in this study may help to select other candidates for more indepth studies aimed at unravelling the role of LecRKs in various biological processes, in stress responses or in interaction with pathogens.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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Supplementary data are available at JXB online.
Supplementary Table S1 contains the MPSS 17 bp tag expression data of the Arabidopsis LecRK genes.
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Acknowledgements |
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We would like to acknowledge Michael Seidl and Ronnie de Jonge for insightful discussions and Dr Harold Meijer for constructive feedback on the first manuscript drafts. This research was supported by the Dutch Ministry of Agriculture, Nature and Food quality, LNV427 grant (‘Parapluplan Phytophthora’) and by an EU-BioExploit grant (FOOD-CT-2005-513959).
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RLK, receptor-like kinase; MAMP, microbe-associated molecular pattern; CW, cell wall; PM, plasma membrane; WAK, wall-associated kinase; PERK, proline-rich extensin-like receptor kinase; GNA, Galanthus nivalis agglutinin; SI, self-incompatibility; SRK, S-locus receptor kinase; EGF, epidermal growth factor; PAN, plasminogen-apple-nematode; LecRK, legume-like lectin receptor kinase; RGD, arginine-glycine-aspartic acid; JAK, Janus kinase; MPSS, massively parallel signature sequencing; LPS, lipo-polysaccharides; NN, neural networks; HMM, hidden Markov models.
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsSupplementary dataReferences |
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