JXB Advance Access originally published online on February 23, 2007
Journal of Experimental Botany 2007 58(6):1381-1396; doi:10.1093/jxb/erl304
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RESEARCH PAPER |
Receptor-like protein kinase HvLysMR1 of barley (Hordeum vulgare L.) is induced during leaf senescence and heavy metal stress
1Martin-Luther Universität Halle-Wittenberg, Institut für Biologie, Weinbergweg 10, D-06120 Halle, Germany
2Friedrich-Schiller-Universität Jena, Institut für Allgemeine Botanik, Am Planetarium 1, D-07743 Jena, Germany
* To whom correspondence should be addressed. E-mail: klaus.humbeck{at}pflanzenphys.uni-halle.de
Received 19 September 2006; Revised 12 December 2006 Accepted 18 December 2006
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Abstract |
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The Hordeum vulgare cDNA clone HvLysMR1 that encodes a putative receptor-like protein kinase was identified by restriction fragment differential display–polymerase chain reaction (PCR) comparing cDNA populations derived from mRNAs of primary leaves stressed with chromium for 48 h with controls. The full-length sequence codes for a protein with 622 amino acids which includes characteristic domains of lysine motif receptor-like kinases: an N-terminal signal peptide, two lysine motifs, a transmembrane region, and a serine/threonine kinase domain at the C-terminal end. The expression of HvLysMR1 is induced during exposure to different heavy metals and its transcript accumulates during leaf senescence. Addition of the calcium ionophore A23187 [GenBank] induces HvLysMR1 expression, indicating the involvement of Ca2+ in the regulation of HvLysMR1. In vitro phosphorylation of HvLysMR1 was analysed with [32P]ATP. Using the overexpressed and purified HvLysMR1–kinase domain, the phosphorylation of HvLysMR1 could be confirmed by nano-liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI–MS) with neutral loss-triggered MS–MS–MS spectra at amino acids localized at the juxtamembrane region. The involvement of HvLysMR1 during heavy metal stress and leaf senescence is discussed.
Key words: Calcium, heavy metal stress, Hordeum vulgare, leaf senescence, lysine motif, receptor-like kinase
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Introduction |
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In the past few years, responses of plants to heavy metals have received increasing attention. On one hand, due to industrial activities, toxic heavy metals such as chromium have been released into the biosphere and represent a widespread environmental pollutant. Plants, like other organisms, have evolved various protective mechanisms against harmful effects of such heavy metals (Hall, 2002). The high potential of some plants to accumulate heavy metals has gained interest for phytoremediation technologies. On the other hand, some heavy metals are essential micronutrients fulfilling many crucial functions in plant metabolism. Understanding the mechanisms by which metals, both essential and non-essential, can be taken up, transported, and incorporated into their target protein, and also sequestered, stored, and detoxified in various organisms may contribute to the optimization of phytoremediation processes (Clemens et al., 2002).
In the past few years, substantial progress has been made in elucidating the mechanistic basis of the homeostasis and detoxification of metals and metalloids in plants (Krämer, 2005), but we are still far away from understanding the regulatory network underlying these processes. Interestingly, several heavy metal-induced genes are also up-regulated during leaf senescence (Himelblau et al., 1998; Himelblau and Amasino, 2000; Guo et al., 2003). Senescence is a complex and highly regulated process that occurs as part of plant development or can be prematurely induced by stress (Buchanan-Wollaston et al., 2003). Leaf senescence involves mobilization of nutrients released after catabolism of macromolecules, including heavy metals such as Cu and Zn (Himelblau and Amasino, 2001). In addition, heavy metal stress often induces senescence-like degradation processes in plants (Chen and Kao, 1999; McCarthy et al., 2001). This indicates an overlap in the regulatory mechanisms underlying heavy metal homeostasis and leaf senescence. One common early event in both processes, heavy metal stress and leaf senescence, is the accumulation of reactive oxygen species (ROS) such as O2·–, H2O2, and ·OH (Krupinska et al., 2003; Mithöfer et al., 2004). These ROS induce oxidative damage in biomolecules and have been proposed to function as signals in stress response and development (Mittler et al., 2004). Another central stress signalling molecule in plants also associated with ROS is Ca2+ (Reddy, 2001; Sanders et al., 2002).
Receptor-like protein kinases are thought to play key roles in the perception and transduction of extracellular signals. They have been shown to be involved in cellular signalling pathways in plant development, disease resistance, or self-incompatibility (Baudino et al., 2001). Here, the characterization of a novel barley (Hordeum vulgare L.) receptor-like kinase, which is induced after exposure to different heavy metals and during the phase of leaf senescence, and also in response to changes in Ca2+ levels, is reported. The derived protein exhibits the typical structure with a signal peptide at the N-terminus, two lysine motifs, a transmembrane domain, and an active intracellular kinase domain (KD).
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Materials and methods |
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Plant material
Barley (Hordeum vulgare L. cv. Steffi) was used in this study. Seedlings were grown hydroponically on Murashige and Skoog medium (Duchefa Biochemie BV, The Netherlands) for 7 d under controlled growth chamber conditions [16 h, 21 °C, and 100 mmol m–2 s–1 PAR (photosynthetic active radiation: 400–700 nm); 8 h, 16 °C, and darkness], and then treated with 50 µM or 1 mM potassium dichromate (K2Cr2O7), cadmium chloride (CdCl2), or copper chloride (CuCl2) for different times, or not treated (controls). For analysis of age-dependent expression of HvLysMR1, plants were grown for 9, 26, and 38 d at 16 h light (21 °C and 100 mmol m–2 s–1 PAR, 400–700 nm) and 8 h darkness (16 °C) on soil containing 4 g of fertilizer (Osmocote 5M; Urania, Hamburg, Germany) per litre of soil. In order to analyse the effects of the calcium ionophore A23187 [GenBank] on expression of HvLysMR1, primary leaves of 7-d-old barley plants grown on Murashige and Skoog medium were cut and then immersed in water containing 200 µM calcium ionophore A23187 [GenBank] for 5, 10, 24, and 48 h. For application of the calcium ionophore A23187 [GenBank] (Sigma-Aldrich, Germany), a stock solution of 1 mM was prepared in dimethylsulphoxide (Roth, Karlsruhe, Germany) and then dissolved in distilled water by rapid mixing. Controls were treated in the same way except for addition of the calcium ionophore. In order to analyse the effects of ROS, barley plants were grown hydroponically as described and then 50 µM methylviologen in 0.1% (v/v) Tween-20 was sprayed onto the leaves. Control plants were treated only with 0.1% (v/v) Tween-20. After an incubation of 1 h in the dark, for improved uptake of the herbicide, plants were exposed to a PAR of 300 mmol m–2 s–1 to induce accumulation of ROS. Samples of leaves were harvested at an appropriate time point, immediately frozen in liquid nitrogen, and stored at –80 °C until use.
Chlorophyll content
Relative chlorophyll content per unit leaf was determined in the middle region of intact leaves during heavy metal treatment using a soil plant analysis development (SPAD) analyser (Minolta, by Hydro Agri, Dülmen, Germany) which measures transmission of wavelengths absorbed by chlorophylls in intact leaves. Each data point represents the mean of 10 independent measurements.
Photosystem II (PSII) efficiency
Chlorophyll fluorescence measurements were performed in the middle region of intact leaves after dark adaptation as described by Humbeck et al. (1996) using a chlorophyll fluorometer (Mini PAM; Walz, Effeltrich, Germany). Mean values of the ratio variable fluorescence/maximal fluorescence (Fv/Fm) are based on 10 independent measurements.
RNA isolation
Total RNA was extracted from leaf tissue using the method of Chirgwin et al. (1979) and was quantified spectrophotometrically. To verify quality of RNA, 10 mg of total RNA was fractionated on a 1% (w/v) agarose gel containing 4% formaldehyde, stained with ethidium bromide, and then visualized under UV light. For northern analysis, RNA was electrophoretically fractionated on 1% (w/v) agarose gels containing 4% formaldehyde. The RNA was transferred by pressure blotting (Stratagene) onto positively charged nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). The membranes were pre-hybridized at 50 °C for 1.5 h in a pre-hybridization solution consisting of 50% (v/v) deionized formamide, 74.7 mM sodium citrate, pH 7.0, 747 mM sodium chloride, 50 mM sodium phosphate, 2% (w/v) blocking reagent (Roche Diagnostics GmbH), 0.1% (w/v) N-lauroylsarcosine, and 7% (w/v) SDS. The hybridization was carried out overnight at 50 °C in a solution containing the same ingredients as the pre-hybridization solution plus the digoxigenin (DIG)-labelled probe prepared from the cDNA fragments isolated by restriction fragment differential display–polymerase chain reaction (RFDD–PCR) using DIG-labelled dUTP (‘Dig-High-Prime’-Kit; Roche Diagnostics GmbH).
RFDD-PCR
The RFDD-PCR was performed according to the instructions of the displayProfile Expression Profiling Kit (Qbiogene GmbH, Heidelberg, Germany). The poly(A)+ RNA was isolated from total RNA using the PolyATtract mRNA Isolation system IV of Promega (Madison, WI, USA). cDNA was synthesized, digested with TaqI enzyme, and ligation of adaptors and final PCR was performed with 32P-labelled primers.
The population of cDNA fragments of each sample was loaded on an 8% (w/v) denaturing polyacrylamide gel and autoradiographed with Kodak Biomax MR-film (Eastman Kodak, Rochester, NY, USA). The cDNA fragments differentially expressed were eluted by boiling the gel pieces in 10 mM TRIS–1 mM EDTA for 10 min at 95 °C, re-amplified by PCR with the same pair of primers as used for the first amplification, cloned by using the pGEM-T® Vector System I (Promega), and sequenced.
cDNA amplification of HvLysMR1
The RFDD-PCR clone HvLysMR1 identified (183 bp) was compared with other barley expressed sequence tags (ESTs) using the HarvEST Triticeae software version 0.99 (University of California, USA). It shows 156/185 bp identities to the HarvEST consensus performed with the AV946685
[GenBank]
barley EST. The cDNA was isolated by first reverse transcription–PCR (RT–PCR) using the OneStep RT–PCR Kit (Qiagen, Hilden, Germany) and the gene-specific primers forward (5'-GAC GCT TTG CTT CCA TCT TCT GA) and reverse (5'-CCT TTG CAC GGG TGT TCT TGT CTA TC), then extended by a second RT–PCR using primers forward (5'-CAT TCA TGA GCA TAC CGT TCC AGT GTA CAT) and reverse (5'-AGA CGA GGT CAT CTC CCG GAC ATT AGG TTC).
The total cDNA cloned by RT–PCR was 992 bp. The full-length sequence of cDNA clone HvLysMR1 was investigated using the GeneRacerTM (Invitrogen, Karlsruhe, Germany) and the gene-specific primers 5' Race (5'-GGG TCT TGA GTA CAT TCA TGA GCA TAC CGT TCC AGT G), Nest 5' Race (5'-CCG CAA ACA TCT TGA TAG ACA AAA CAC CCG TGC AAG G), 3' Race (5'-CCG AAG CTG GGA GAC GAC TAT CCT GTC GAT GC), and Nest 3' Race (5'-CTC ATG ATG ACG CAC CTG GCG AAC GCA TGC AC). PCR was performed with poly(A)+ RNA isolated from the total RNA sample extracted from primary barley leaves that were treated with chromium for 48 h. The PCR products were cloned by using the pGEM-T® Vector System I (Promega) and sequenced.
Sequencing analysis
Nucleotide sequences were determined by the dideoxy chain termination method using the BigDye® Terminator v1.1 Cycler Sequencing Kit (Applied Biosystems, Forster City, CA, USA) with a sequencer ABI PrismTM 370 automatic DNA Sequencer (Applied Biosystems), analysed using the Lasergene expert sequence analysis software (DNASTAR Inc., Madison, WI, USA), and compared with the DNA and proteins using the EMBL FASTA server (Pearson and Lipman, 1988), the BLAST server at NCBI (Altschul et al., 1990), and HarvEST Triticeae software version 0.99 (University of California, USA). The primers NewT7 5'-GTA ATA CGA CTC ACT ATA GGG C and SP6 5'-AGC TAT TTA GGT GAC ACT ATA G were used for sequencing of cDNA fragments cloned into the pGEM-T® Vector.
Quantitative real-time PCR
Total RNA was treated with DNase I, and cDNA was synthesized using the Omniscript Reverse Transcriptase Kit (Qiagen, Hilden, Germany). PCR was carried out in an iCycler (BioRad, Munich, Germany) in a total of 25 µl using 1x SYBR Green I fluorescence dye that binds to the double-stranded DNA, 5 µM of the gene-specific primers, forward primer 5'-CAA CGT GAA CGT CTC CTA CAT CGC ATC G, reverse primer 5'-GGC AGC GTG AGG CAC TTG CAT GTG A, and the 18S rRNA housekeeping gene primers, forward primer 5'-CAG GTC CAG ACA TAG CAA GGA TTG ACA G and reverse primer 5'-TAA GAA GCT AGC TGC GGA GGG ATG G with different dilution of cDNA 1/4, 1/16, and 1/64. To test the specificity of RT–PCR products of target and reference genes, they were separated in a 1% (w/v) agarose gel which always resulted in single products, and then cloned and sequenced. To determine the relative quantification based on the relative expression of a target gene versus a reference gene, a relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR described by Pfaffl et al. (2002) was used.
Overexpression of His-HvLysMR1–KD
The KD of HvLysMR1 was amplified by PCR from the original HvLysMR1 plasmid and primers (forward) 5'-GAC ATA TGA GGC GAA GAA AGG CGA AAC AGG GTG with an NdeI site and (reverse) 5'-GAC TCG AGT CAT CTC CCG GAC ATG AGG TTC ACC A with an XhoI site. The PCR product was ligated into the NdeI/XhoI site of a pET-15b vector (Novagen, Darmstadt, Germany) to produce a recombinant His-HvLysMR1–KD protein.
The recombinant plasmid was introduced into Escherichia coli Rosetta (DE 3) pLys S (Novagen, Darmstadt, Germany), and overexpression of protein was induced by addition of 0.2 mM isopropyl ß-D-thiogalactopyranoside (IPTG; Duchefa, Biochemie BV) to the medium. After incubation at 37 °C for 1 h, bacterial cultures were harvested and resuspended in phosphate-buffered saline (PBS) [150 mM NaCl, 16 mM Na2 HPO4, 4 mM KH2 PO4, 2% (v/v) Triton X-100] before lysis by sonification. The recombinant protein His-HvLysMR1–KD was purified in an Ni-NTA Superflow Column (1.5 ml) (Qiagen, Hilden, Germany).
Western blot analysis
The overexpressed protein was separated by SDS–PAGE and transferred onto a polyvinylidene difluoride (PVDF) WESTRAN® CLEAR SIGNAL 0.45 µm membrane (Schleicher & Schuell, Dassel, Germany) by electroblotting. The membrane was incubated in the first antibody (Penta Anti-His; Qiagen, Hilden, Germany) for 1 h at room temperature and then in horseradish peroxidase-conjugated second antibody (anti-rabbit; Amersham, Freiburg, Germany). For detection, the ECL Plus Western blotting detection reagents (Amersham Biosciences, UK) were used. The membrane was then exposed on a film (HyperfilmTM ECLTM, Amersham Pharmacia Biotech Europe, Freiburg, Germany) for development.
Autophosphorylation analysis
The His-tag fusion protein HvLysMR1–KD was analysed for its kinase activity. Phosphorylation was carried out in 25 µl volumes of assay buffer [50 mM TRIS–HCl, pH 7.6; 50 mM KCl, 2 mM dithiothreitol (DTT), 10% (v/v) glycerol] each containing 0.5 µg of His-tag fusion protein in the presence of 5 mM MgCl2 and 5 mM CaCl2. The phosphorylation reaction was initiated by adding 12.5 µCi of [
-32P]ATP (Amersham Biosciences, Freiburg, Germany) or without (control), then samples were incubated at 22 °C for 45 min. The reaction was stopped by addition of EDTA to a final concentration of 10 mM. The phosphorylated protein was analysed using 15% SDS–PAGE and transferred onto a WESTRAN® CLEAR SIGNAL 0.45 µm PVDF membrane (Schleicher & Schuell, Dassel, Germany). The incorporated phosphate was visualized by exposure of the membrane to Imaging Plate for 2 d and analysed using a Fluorescent Image Analyser (FLA-3000 Series; Fuji Photo Film Co., Tokyo, Japan).
Peptide identification by nano-liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI–MS) (MS2 and neutral loss-triggered MS3)
A 20 µl aliquot of the eluate of the overexpressed His-HvLysMR1–KD that was released from the Ni-NTA Superflow Column was digested with 20 ng of trypsin (Promega, Madison, WI, USA) at 37 °C overnight following the protocol of Promega. The peptide solution was desalted with ZipTips according to Wagner et al. (2006) and vacuum dried. The resulting pellet was dissolved in 5 µl of buffer A (see below), and the peptides were subjected to nano-LC-ESI-MS analysis using a nanoscale C18 column (flow rate, 300 nl min–1) coupled online to a linear ion trap mass spectrometer (Finnigan LTQ; Thermo Electron Corp., San Jose, CA, USA). The LC system consisted of a Dionex UltiMate3000 liquid chromatography system (Dionex), including an autosampler, a flow control module, and a micropump. A gradient was used to elute peptides from the reversed-phase column [length, 15 cm; inner diameter, 75 µm; C18Pepmap100 particle size, 3 µm (Dionex P/N 160321)].
The successive steps of the applied gradient were as follows: 5 min, 96% (v/v) A–4% (v/v) B; 30 min, gradually shifting to 50% (v/v) A–50% (v/v) B; 5 min, gradually shifting to 10% (v/v) A–90% (v/v) B; 10 min, 10% (v/v) A–90% (v/v) B; and 1 min, gradually shifting to 96% (v/v) A–4% (v/v) B, where A consists of 5% (v/v) acetonitrile, 0.1% (v/v) formic acid in water, and B consists of 80% (v/v) acetonitrile, 0.1% (v/v) formic acid in water.
The instrument was run in the data-dependent neutral-loss mode, cycling between one full MS scan and MS2 scans of the four most abundant ions. The detection of a neutral loss fragment (196, 98, 49, or 32.66 Da) in the MS2 scans immediately triggered an MS3 scan of the precursor ion representing the dephosphorylated peptide. The analysis of the resulting spectra was done according to Wagner et al. (2006). The MS2 and MS3 data were used to search the database with the protein sequence of the overexpressed protein using Bioworks software (version 3.2; Thermo Electron Corp., San Jose, CA, USA) including the SEQUEST algorithm (Link et al., 1999). The software parameters were set to detect a modification of 79.96 Da in serine, threonine, or tyrosine in MS2 and MS3 spectra. When phosphoserine and phosphothreonine undergo gas-phase ß elimination, dehydroalanine (Dha) and 2-aminodehydrobutyric acid [methyldehydroalanine (MeDha)], respectively, are produced. Thus, modifications of –18.00 Da in serine and threonine residues were additionally used for database searches with MS3 data. Searches were done for tryptic peptides, allowing two missed cleavages. Mass tolerance was set to 1.5 Da for the peptide precursor ion in MS mode. For fragment ions (MS2 and MS3 modes), mass tolerance was set to 1.0 Da. Scores for the Xcorr factor (Eng et al., 1994) were set to the following limits: Xcorr of >1.5 if the charge of the peptide was 1, Xcorr of >2 if the charge of the peptide was 2, and Xcorr of >2.5 if the charge of the peptide was 3.
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Isolation of a cDNA encoding a lysine motif receptor-like kinase from chromium-stressed barley leaves
An RFDD-PCR experiment was carried out in order to identify genes that are induced during chromium treatment in barley primary leaves. Equal amounts of cDNA were synthesized from mRNAs isolated from primary leaves of barley seedlings grown hydroponically on Murashige and Skoog medium for 7 d and then treated with 1 mM potassium dichromate (K2Cr2O7) for 48 h or not (control). Differences in the populations of cDNAs in these two samples were compared on 8% (w/v) polyacrylamide gels using the differential display approach. In total, 48 cDNA fragments of genes differentially expressed during the first phase of chromium treatment could be detected. Seven fragments were re-amplified by PCR and sequenced. cDNA sequences were compared with those present in the GenBank databases. These cDNA clones up-regulated during chromium treatment were named chromi 1–7 (chromium-induced gene) (Table 1). Four clones were shown by northern analysis to be up-regulated upon chromium stress (Fig. 1A). For the clones whose mRNA could not be detected by the northern technique (data not shown), the transcript levels were analysed via quantitative real-time PCR (Fig. 1B). The 183 bp fragment (chromi 1, AJ630116 [GenBank] ) showing homology to a receptor-like kinase was characterized in more detail in this report. The identified clone was compared with other barley ESTs using the HarvEST Triticeae software version 0.99 (University of California, USA) showing 156/185 bp identity to the HarvEST consensus performed with the AV946685 [GenBank] barley EST. First, 993 bp of this cDNA clone were amplified by RT–PCR and sequenced. The full-length sequence including 5' and 3' ends of this cDNA clone was obtained by using a RACE (rapid amplification of cDNA ends) technique. The full-length cDNA comprises 2119 bp including an open reading frame of 1868 bp (nucleotides 48–1916; CAJ14969 [GenBank] ) coding for 622 amino acids from Met1 to Arg622 (Fig. 2A). By comparison of the deduced amino acid sequence, a LysM motif from amino acids His110 to Pro148 and Phe176 to Pro217 could be identified, showing homology to the consensus sequence LysM domain (PF01476) available at www.sanger.ac.uk/Software/Pfam/search.shtml (Fig. 2C). The LysM protein module is found among both prokaryotes and eukaryotes, and was first identified in bacterial lysine and muramidase enzymes that degrade cell wall peptidoglycan (Radutoiu et al., 2003). In addition to LysM motifs (at the extracellular region of the receptor-like kinase), the identified HvLysMR1 presents an N-terminal signal peptide from amino acids Met1 to Ala27, a transmembrane domain from amino acids Ala240 to Tyr262 identified using CBS analyses (Center for Biological Sequence Analysis from Technical University of Denmark), and a KD from amino acids Phe327 to Val594 with 11 characteristic subdomains of protein kinases (Radutoiu et al., 2003) (Fig. 2A, B). Due to these characteristic domains, the gene was named HvLysMR1 (Hordeum vulgare lysine motif receptor-like kinase 1). Alignment of the novel barley HvLysMR1 reveals homologies to others plant LysM receptor-like kinases identified in Medicago truncatula (AAQ73157 [GenBank] , AAQ73154 [GenBank] , AAQ73160 [GenBank] , AAQ73155 [GenBank] , and AAQ73158 [GenBank] ), Lotus japonicus (CAE02589 [GenBank] and CAE02597 [GenBank] ), and Pisum sativum (SyM 10) (Fig. 2C). Figure 2D shows the phylogenetic tree of these genes and of further genes with as yet unknown functions from Arabidopsis thaliana (At BAB02358 [GenBank] , At NP_56689; At AAB80675 [GenBank] ; At NP_180916 [GenBank] ). Receptor-like kinases with the lysine motif have, so far, been found only in plants, and some of them are described to play a role in recognition of symbiotic bacteria in the legume plants L. japonicus (Radutoiu et al., 2003) and M. truncatula (Limpes et al., 2003).
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HvLysMR1 is induced during chromium, cadmium, and copper treatment
Expression patterns of the newly identified HvLysMR1 in the primary leaves of barley were analysed using high (1 mM, fast response) and low (50 µM, slow response) concentrations of chromium, cadmium, and copper. To characterize the stress response of the primary leaves physiologically under these conditions, two photosynthesis-related stress parameters were measured: chlorophyll content and PSII efficiency. The chlorophyll content of primary leaves significantly decreased during exposure to 1 mM potassium dichromate within the first 48 h (Fig. 3B). After 96 h chromium treatment, only 60% of chlorophyll of control is left in the leaves. This indicates a stress-dependent chlorophyll degradation during chromium exposure. The second photosynthesis-related stress parameter, PSII efficiency, was measured after dark adaptation as described by Humbeck et al. (1996). This sensitive parameter is decreased already after 24 h of this chromium treatment.
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Changes in mRNA levels of HvLysMR1 during exposure to 1 mM chromium were investigated using quantitative real-time PCR. Transcript levels of HvLysMR1 in the leaves were compared with those of the reference gene (18S rRNA). mRNA levels in controls were always set as 1 at the different time points (Fig. 3A). HvLysMR1 expression in the leaves was already slightly induced during the first 10 h of 1 mM chromium treatment and maximum transcript levels were detected after 24 h of treatment (10.5 times higher than in the control; Fig. 3A). After this time point, mRNA levels decrease again during prolonged stress times. This result indicates a transient expression pattern of HvLysMR1 during chromium stress.
In order to investigate whether the expression of HvLysMR1 is also affected by heavy metals other than chromium, 7-d-old barley seedlings were treated with either 1 mM cadmium chloride or 1 mM copper chloride for 24, 48, 72, and 96 h, or not treated (controls). In addition, changes in the two photosynthesis-related stress parameters, chlorophyll content and PSII efficiency, were measured in the two experiments (Figs 4B and 5B). In cadmium-treated seedlings, chlorophyll content in the leaves starts to decrease 24 h after onset of cadmium treatment. This decrease is accentuated during prolonged time of exposure. PSII efficiency decreased at 48 h of cadmium treatment. In seedlings treated with copper (Fig. 5B), patterns of changes in chlorophyll content and PSII efficiency similar to those during cadmium treatment were observed (Fig. 4B).
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The expression levels of HvLysMR1 under cadmium and copper treatment were again investigated via quantitative real-time PCR in comparison with the controls. During cadmium treatment, after 24 h of exposure, the mRNA level of HvLysMR1 is significantly increased and high levels of this transcript could be identified 48 h after onset of the treatment (147.4 times more than in controls, Fig. 4A). Treatment with 1 mM copper also resulted in increased mRNA levels of HvLysMR1 already 24 h after onset of chromium treatment (3.4 times more than in controls, Fig. 5A). After this time point, the relative mRNA levels of HvLysMR1 declined to reach basal levels at 48 h. These data indicate a fast and transient expression pattern after exposure of the plant to high concentrations of the essential heavy metal copper and the non-essential heavy metal cadmium.
After exposure to low concentrations (50 µM) of chromium and cadmium, the stress parameters chlorophyll content and PSII efficiency do not change significantly in the leaves during 72 h (Fig. 6B). Treatment with 50 µM copper results in a slight decrease in both parameters after 48 h (Fig. 6B).
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The effect on HvLysMR1 mRNA levels in these leaves was investigated again via quantitative real-time PCR by comparison of mRNA levels of the HvLysMR1 gene with the levels of the reference 18S rRNA from treated samples and controls at different time points. Treatment with 50 µM copper resulted in a clear increase in the HvLysMR1 transcript level already after 48 h (6.4 times higher than in control; Fig. 6A). In later stages, mRNA levels decreased again. These results indicate a transient expression pattern of HvLysMR1 during copper treatment. Low concentrations of chromium and cadmium (50 µM) caused a slight but significant increase in HvLysMR1 mRNA level after 72 h (2.1 times higher in the chromium-treated seedlings and 1.8 times higher in the cadmium-treated seedlings compared with the control, Fig. 6A). The fast response of HvLysMR1 to low concentrations of the essential heavy metal copper could be explained by the fact that copper is efficiently taken up by specific transporters (Aller et al., 2004), while the non-essential heavy metal cadmium was shown to be transiently retained in the root system and only slowly transported to the shoot (Page and Feller, 2005). Cadmium is taken up into plant cells, most probably via Ca2+, Fe2+, and Zn2+ uptake systems such as LCT1 that mediates both Ca2+ and Cd2+ transport into the cytosol of cells (Clemens et al., 1998). Since plants lack a specific transport system for chromium, it is taken up by carriers of essential ions such as sulphate or iron (Shanker et al., 2005) and predominantly accumulates in the roots, while only low concentrations are transported to the shoots (Han et al., 2004).
HvLysMR1 mRNA accumulates during leaf senescence
Some heavy metal-induced genes are also up-regulated during leaf senescence (Himelblau et al., 1998; Himelblau and Amasino, 2000; Guo et al., 2003), indicating an overlap in the response of plants to these two conditions. For this, tests were conducted to determine whether the newly identified receptor-like kinase is also induced during leaf senescence. Barley plants were grown for 9, 26, and 38 d as described in Materials and methods. At 9 d after sowing, primary leaves are in their mature stage. After 26 d and 38 d, these leaves are in the early and late stages of senescence. Figure 7 shows that the relative HvLysMR1 expression rate significantly increases during leaf senescence, showing 15–19 times higher values during senescence than in the mature leaf. Levels of the 18S rRNA reference during senescence do not differ more than 15%.
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HvLysMR1 mRNA level responds to addition of the calcium ionophore A23187 and methylviologen treatment
The two other known LysM receptor-like kinases Lj NFR5 and Lj NFR1 have been shown to be involved in Nod factor signal transduction which implies Ca2+ signalling (Oldroyd and Downie, 2004). Such changes in cytosolic calcium play a central role during the transduction of a wide variety of abiotic and biotic signals and in growth and developmental processes (Reddy, 2001; Sanders et al., 2002), and it is known that many stress-related genes are regulated in response to intracellular calcium levels (Albrecht et al., 2003). In order to investigate whether changes in cytosolic calcium could also be involved in the regulation of the newly identified LysM motif receptor-like kinase HvLysMR1, an experiment was performed with the calcium ionophore A23187 [GenBank] , which is reported to induce changes in cytosolic calcium concentration (Peiretti et al., 1997; Kim et al., 2003). A fast and significant accumulation of HvLysMR1 mRNA was observed already 5 h after addition of the calcium ionophore A23187 [GenBank] (Fig. 8A). In later stages of the treatment, the mRNA levels decrease again.
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Another common signal in response to different stress conditions including heavy metal treatment and also during onset of senescence is the accumulation of ROS (Krupinska et al., 2003; Mithöfer et al., 2004). Changes in amounts of ROS were also described to play a role in Ca2+ signalling (Himmelbach et al., 2003). In order to verify whether ROS are involved in the regulation of HvLysMR1, methylviologen was used as a source which generates superoxide anion radicals by uptake of an electron from PSI (Donahue et al., 1997). Seven-day-old barley plants hydroponically cultivated on Murashige and Skoog medium were sprayed with 50 µM methylviologen in 0.1% (v/v) Tween-20, then incubated for 1 h in the dark for improved uptake of methyviologen. Plants were then exposed to a PAR (400–700 nm) of 300 mmol m–2 s–1 to induce oxidative stress for 1.5, 3, and 6 h. The control was treated only with 0.1% (v/v) Tween-20. The results obtained in Fig. 8B show that methylviologen causes a very slight but significant accumulation of HvLysMR1 mRNA.
The HvLysMR1 intracellular domain encodes a functional kinase
It is known that the intracellular part of receptor-like kinases undergoes autophosphorylation which plays a role in signal transduction (Yoshida and Parniske, 2005). In order to investigate whether the newly identified HvLysMR1 protein possesses such an active KD, the His-tagged HvLysMR1–KD (Fig. 9A) was overexpressed in Escherichia coli, purified using a Ni-NTA Superflow Column, and immunologically analysed with an anti-His antibody (Qiagen, Hilden, Germany). Figure 9B shows a band of 
42 kDa corresponding to the molecular mass of the overexpressed protein. Autophosphorylation was tested by incubation of the purified protein with [-32P]ATP. The phosphorylated protein was analysed after SDS–PAGE, transfered onto a PVDF membrane, and visualized by autoradiography (Fig. 9C). The results prove a weak phosphorylation of a protein showing a similar molecular mass to the immunologically identified chimeric protein (Fig. 9B). The weak phosphorylation indicates that the overexpressed protein was already phosphorylated to a great extent in E. coli. In order to prove that the intracellular domain of HvLysMR1 can indeed be phosphorylated as known for active receptor-like kinases, the overexpressed protein was additionally analysed using nano LC-ESI-MS by MS2 and MS3 spectra using the data-dependent neutral loss mode (Wagner et al., 2006). Thus, neutral loss events (loss of phosphoric acid from a phosphorylated serine or threonine resulting in a Dha or MeDha) that occurred during MS/MS are used to trigger an MS3 automatically. MS analysis was carried out in two experiments with independent eluates from the His-HvLysMR1–KD overexpression. It revealed 13 different peptides with significant Xcorr (Table 2), giving an amino acid coverage of 47.8%. From the detected peptides, only one (LASTILIQK) could be identified as a phosphopeptide. However, it was also present in its non-phosphorylated form, indicating that only a portion of the protein is phosphorylated. The phosphopeptide LASpTILIQK was found with Dha instead of Ser284, indicating a neutral loss on the phosphorylated side chain of serine. It was possible to assign positively the complete y-ion series from this peptide and all b-ions with a mass >300 m/z, resulting in a clear identification of this phosphopeptide (Fig. 10, Table 2). In addition, the same peptide with phosphorylation at the threonine residue (LASTpILIQK) was found; however, only with a relatively low abundant y-ion at the relevant position. The experiments show that either Ser284 or to a lesser extent Thr285 within peptide LASTILIQK that is located at the juxtamembrane region is phosphorylated. Earlier studies of phosphorylation sites of plant receptor-like kinases revealed that phosphorylation at this juxtamembrane region is a common feature (Nühse et al., 2004; Wang et al., 2005).
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cDNA populations derived from chromium-treated barley plants were compared with those of control plants via RFDD-PCR. The cDNA clone HvLysMR1 which was isolated by this approach was investigated in more detail in the present report. The derived amino acid sequence exhibits at the N-terminal end a hydrophobic stretch of 27 amino acids which is predicted to act as a signal peptide for the secretory pathway by the TargetP 1.1 Server from CBS analysis (Center for Biological Sequence Analysis from Technical University of Denmark). Furthermore, the sequence shows two LysM motifs which are typical for lysine motif receptor-like kinases (Limpens, 2003; Madsen et al., 2003), a hydrophobic membrane-spanning segment, and a conserved serine/threonine KD with 11 characteristic subdomains of protein kinases (Radutoiu et al., 2003). This derived structure strongly suggests that the encoded protein is a lysine motif receptor-like kinase with an extracellular LysM part responsible for perception of incoming extracellular signals, which is fixed by its hydrophobic membrane-spanning segment at the plasma membrane and transduces the signal to intracellular signalling pathways via its intracellular KD (Fig. 2). The novel HvLysMR1 exhibits several motifs that are highly conserved in the protein kinase superfamily. The known Asp-Phe-Gly (DFG) motif in the subdomain VII (Fig. 2A) is suggested to chelate Mg2+ ions required for autophosphorylation activity (Nishiguchi et al., 2002). Another conserved subdomain VIII, which is assumed to be involved in the recognition of the substrates, consists of an Ala-Pro-Glu (APE) motif (Nishiguchi et al., 2002). In HvLysMR1, the alanine of this motif is replaced by proline. Such a divergence in this activation loop (subdomain VIII) or even the complete absence of the activation loop in the KD of the lysine motif receptor-like kinase NFR5 from L. japonicus was already discussed by Madsen et al. (2003). Alterations in the conserved subdomains of the KD were also reported for other serine/threonine receptor-like kinases from pea, Medicago, and rice. They lack the aspartic acid residue in domain VII, and the activation loop in domain VIII is highly diverged or absent (Madsen et al., 2003).
Plant receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein-coding genes, and most of them described in plants, so far, encode serine/threonine kinases (Shiu and Bleecker, 2001). They fulfil fundamental functions in the perception and processing of various extracellular signals via cell surface receptors and, according to their divergent extracellular receptor domains, can be grouped into 15 different subfamilies (Shiu and Bleecker, 2001, 2003). This divergence allows them to respond to a wide range of external signals. The extracellular domain of the novel HvLysMR1 protein contains two LysM motifs showing homologies to the conserved LysM domain (PF01476) of plant receptor-like kinases and amino acid sequences of LysM motifs of known LysM receptor-like kinases (Fig. 2C). For this reason, the newly identified receptor-like kinase was classified as being a member of the LysM receptor-like kinase subfamily and named HvLysMR1.
Recently, plant LysM receptor-like kinases could be shown to be involved in legume perception of rhizobial signals (Limpens et al., 2003; Madson et al., 2003; Radutoiu et al., 2003). In RNA interference studies investigating the function of the LysM receptor-like protein kinase LYK3 from M. truncatula, a role in the rhizobia–plant symbiotic process could be proven (Limpes et al., 2003). It was shown that the specific rhizobial signal molecule lipochitin oligosaccharide is detected by plant LysM receptor-like kinases LYK3, NFR1, and NFR2 (Limpens et al., 2003; Madson et al., 2003; Radutoiu et al., 2003; Spaink, 2004). The backbone of this N-acetyl glucosamine Nod factor is similar to that of peptidoglycans known to interact with prokaryotic LysM receptor-like kinases (Riely et al., 2004). The involvement in the signalling process in the legume–rhizobia symbiosis is up to now, as far as we know, the only clear functional assignment of LysM plant receptor-like kinases. So far, there are no other reports about other biological functions of plant LysM receptor-like kinases.
In this report, for the first time induction of a LysM receptor-like kinase during heavy metal stress and leaf senescence is shown. Such an overlap in mechanisms involved in heavy metal stress response and leaf senescence is already known. It could be shown that several heavy metal-induced genes are also up-regulated during leaf senescence (Himelblau et al., 1998; Himelblau and Amasino, 2000; Guo et al., 2003). The reason for these overlapping expression patterns might be that during leaf senescence, proteins, including those containing metals, are degraded. The liberated metals have to be sequestered, and a certain amount of these metals is transported to the growing tissues of the plant (Himelblau and Amasino, 2001). Therefore, the same regulatory factors might be involved in the response of plants to both heavy metals and leaf senescence. In addition, senescence processes can be induced either by internal signals such as age or phytohormones or by external stressors (Bleecker and Patterson, 1997; Gan and Amasino, 1997; Kleber-Janke and Krupinska, 1997; Quirino et al., 2000; Humbeck and Krupinska, 2003; Jing et al., 2003). It is known from the literature that heavy metal stress results in degradation processes which partly resemble those observed during leaf senescence (Chen and Kao, 1999; McCarthy et al., 2001). This is also shown by the decrease in chlorophyll content and in PSII efficiency which also occurs in the heavy metal stress experiments (Figs 3–6) as well as during leaf senescence (Miersch et al., 2000).
Two other receptor-like kinases which belong to the leucine-rich repeat receptor kinases subfamily are already known to be induced during senescence: the Phaseolus vulgaris senescence-associated receptor-like kinase (SARK; Hajouj et al., 2000) and the Arabidopsis thaliana senescence-induced receptor-like kinase (At SIRK; Robatzek and Somssich, 2002). However, there are still open questions about their exact functional integration in the complex signalling pathways underlying regulation of leaf senescence. To our knowledge, up to now no receptor-like kinase is reported to be involved in the heavy metal stress response in plants.
One common early signal inducing both processes, i.e. heavy metal stress and leaf senescence, are ROS, which accumulate during the response to abiotic and biotic stressors (Mithöfer et al., 2004; Rentel and Knight, 2004) and have also been suggested to play a role in the onset of leaf senescence (Krupinska et al., 2003; Mithöfer et al., 2004). A model for an H2O2 signalling pathway was proposed by Hung et al. (2005). Our newly identified HvLysMR1 shows only a very slight induction during the methylviologen treatment, indicating only a minor role, if any, of ROS in induction of this novel LysM receptor-like kinase (Fig. 8B).
Another central messenger involved in regulation of development and stress response is Ca2+ (Reddy, 2001). Aluminium-induced changes in cytosolic Ca2+ concentration have been reported by Lindberg and Strid (1997) and also by Plieth et al. (1999). It is also known that Ca2+ signals are important in nodulation, which also involves the action of LysM receptor-like kinases (as discussed above). It is shown that in root hairs of legumes, nanomolar amounts of Nod factors result in the onset of Ca2+ cytoplasmic spiking (Bothwell and Ng, 2005). In addition, studies show that calcium spiking is essential for Nod factor-induced gene expression (Geurts et al., 2005). Figure 8A shows a fast response of HvLysMR1 to calcium inophore A23187 [GenBank] treatment which is known to enhance Ca2+ influx (Torrecilla et al., 2001). This result might indicate that calcium is involved in the signalling pathways leading to the expression of HvLysMR1.
An essential feature of the function of receptor-like kinases is autophosphorylation of the intracellular part which is required for interaction with downstream regulatory factors in the connected signalling pathways (Robatzek and Somssich, 2002; Yoshida and Parniske, 2005). In order to test whether the novel LysM receptor-like kinase HvLysMR1 is functional in this aspect, autophosphorylation of this protein was analysed using two approaches. On one hand, it could be shown that a protein in the molecular weight range of the overexpressed KD of HvLysMR1 is able to incorporate 32P-labelled phosphate from ATP (Fig. 9C). On the other hand, either Ser284 or, to a lesser extent, Thr285 that are situated in the juxtamembrane region were identified as phosphorylation sites by LC-ESI-MS with neutral loss-triggered MS3 spectra. It cannot be excluded that HvLysMR1 has additional phosphorylation sites which are functionally active. Earlier studies of phosphorylation sites of plant receptor-like kinases revealed that phosphorylation at the juxtamembrane region is a common feature and that there are mult-iple phosphorylation sites responsible for the interaction with downstream signalling factors (Nühse et al., 2004; Wang et al., 2005).
To date, only a few receptor-like kinases have been linked to certain plant processes. These include CLV1 in meristem organization, ERECTA in organ shape, BRI1 in brassinolide signalling, FLS2 in flagellin signalling, HAESA in floral organ abscission, and BrSRK1 in self-incompatibility (Clark et al., 1993; Stein et al., 1996; Torii et al., 1996; Li and Chory, 1997; Gomez-Gomez and Boller, 2000; Shiu and Bleeclker, 2001). The identification in this study of the novel senescence- and heavy metal stress-dependent receptor-like kinase HvLysMR1 is of particular interest, since up to now our knowledge about regulatory components underlying these two processes is very limited. As outlined by Weber et al. (2006), it is not clear whether responses of plants to heavy metals are primarily induced by a direct interaction between the heavy metal and a specific receptor or whether they are induced by signals originating from harmful effects of heavy metals within the cell. The present data showing the involvement of a lysine motif receptor-like kinase in both processes, leaf senescence and heavy metal stress (low and high concentrations), also cannot finally answer this question. HvLysMR1 could either sense on one hand a senescence signal and on the other hand accumulation of heavy metals, or could only interact with a senescence signal which is also elicited by the harmful effects of the accumulating heavy metals. Since this is the first report about a receptor-like kinase induced during heavy metal stress, further functional studies have to provide insight into the molecular mechanisms to understand the role of LysM receptor-like kinase during the plant response to heavy metal stress and the senescence process.
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Acknowledgements |
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We thank Nicole Sommer for providing RNA samples from senescent primary leaves, Wiebke Zschiesche for providing RNA samples from barley plants treated with cadmium and copper, Kathleen Clauss for providing RNA from methylviologen treatment, and Olaf Barth for technical help with sequences alignment. The German Academic Exchange Service (DAAD) and the German Research Foundation (DFG) are thanked for financial support.
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Abbreviations |
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DIG, digoxigenin; EST, expressed sequence tag; KD, kinase domain; LC–ESI–MS, liquid chromatography-electrospray ionization-mass spectrometry; PAR, photosynthetic active radiation; PSII, photosystem II; PVDF, polyvinylidene difluoride; RFDD–PCR, restriction fragment differential display–polymerase chain reaction; ROS, reactive oxygen species; RT–PCR, reverse transcription–polymerase chain reaction.
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