JXB Advance Access originally published online on September 27, 2006
Journal of Experimental Botany 2006 57(14):3767-3779; doi:10.1093/jxb/erl137
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RESEARCH PAPER |
Duplicate maize 13-lipoxygenase genes are differentially regulated by circadian rhythm, cold stress, wounding, pathogen infection, and hormonal treatments
1Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132, USA
2Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August University Göttingen, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany
* To whom correspondence should be addressed. E-mail: kolomiets{at}tamu.edu
Received 12 April 2006; Accepted 25 July 2006
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Abstract |
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Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defence responses to pathogens and pests. By contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. Two maize LOXs, ZmLOX10 and ZmLOX11 that share >90% amino acid sequence identity but are localized on different chromosomes, were cloned and characterized. Phylogenetic analysis revealed that ZmLOX10 and ZmLOX11 cluster together with well-characterized plastidic type 2 linoleate 13-LOXs from diverse plant species. Regio-specificity analysis of recombinant ZmLOX10 protein overexpressed in Escherichia coli proved it to be a linoleate 13-LOX with a pH optimum at
pH 8.0. Both predicted proteins contain putative transit peptides for chloroplast import. ZmLOX10 was preferentially expressed in leaves and was induced in response to wounding, cold stress, defence-related hormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA), and inoculation with an avirulent strain of Cochliobolus carbonum. These data suggested a role for this gene in maize adaptation to abiotic stresses and defence responses against pathogens and pests. ZmLOX11 was preferentially expressed in silks and was induced in leaves only by ABA, indicating its possible involvement in responses to osmotic stress. In leaves, mRNA accumulation of ZmLOX10 is strictly regulated by a circadian rhythm, with maximal expression coinciding temporally with the highest photosynthetic activity. This study reveals the evolutionary divergence of physiological roles for relatively recently duplicated genes. Possible physiological functions of these 13-LOXs are suggested. Key words: Circadian clock, evolution of duplicated genes, green leafy volatiles, jasmonic acid, oxylipins, salicylic acid
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and metabolites derived there from, are important components in plant defence responses to pathogens and pests (Rosahl and Feussner, 2005). Recent inroads in the elucidation of the biological roles of these compounds have occurred in dicot systems. The majority of oxylipins are produced through the lipoxygenase (LOX) pathway, which is comprised of at least seven multienzyme pathway branches (Feussner and Wasternack, 2002). The starting point of the LOX pathway is the incorporation of molecular oxygen into either the 9- or 13-position of the carbon chain of C18 polyunsaturated fatty acids. This reaction is mediated by regio-specific 9- or 13-LOXs that use linoleic (18:2) and linolenic (18:3) acids as substrates. According to the classification based on their primary structure and overall sequence similarity, plant LOXs can also be grouped into two gene subfamilies, type 1 and type 2 LOXs (Shibata et al., 1994). Enzymes designated type 1 have a high sequence similarity (>75%) to one another and lack plastid transit peptide. There are 9- as well as 13-LOXs in this class. However, the type 2 enzymes show relatively low overall sequence similarity (<35%) to one another and carry a putative chloroplast targeting sequence. To date, type 2 LOXs consist exclusively of 13-LOXs (Feussner and Wasternack, 2002). Primary products of LOX enzymatic activity, fatty acid hydroperoxides, are highly reactive and can form free radicals, causing the membrane damage and hypersensitive cell death usually associated with incompatible plant interactions with pathogens (Blée, 2002). However, in healthy cells, LOX-mediated 9- and 13-hydroperoxides are generally quickly transformed into an array of more stable oxylipins with diverse physiological functions (Feussner and Wasternack, 2002; Howe and Schilmiller, 2002).
Products of the 13-LOX enzymatic activity can be utilized by several downstream pathway branches; however, currently the best characterized are the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) pathways. The AOS-mediated or so-called octadecanoid pathway produces jasmonic acid (JA) and its precursors and derivatives with signalling properties, collectively known as jasmonates. It is well established that JA is produced from linolenic acid via sequential enzymatic action of plastid-localized 13-LOX, an AOS, an allene oxide cyclase, and peroxisome-localized 12-oxo-phytodienoic acid reductase (OPR), and three consecutive ß-oxidation steps (Feussner and Wasternack, 2002). Jasmonates function as signals in the pathogen- and insect-induced transduction pathways that regulate expression of defence-related genes (Turner et al., 2002). An antisense-mediated depletion of JA-producing 13-LOXs in Arabidopsis (Bell et al., 1995), potato (Royo et al., 1999), and tobacco (Halitschke and Baldwin, 2003) resulted in increased performance of insect pests. A different set of oxylipins is produced by the chloroplast-localized HPL-mediated pathway. The best studied HPL-derived oxylipins, C6 green leafy volatiles (GLVs), have been shown to possess both antimicrobial (Prost et al., 2005) and defence signalling activities (Bate and Rothstein, 1998). GLVs were shown to enhance the production of anthocyanins in Arabidopsis (Bate and Rothstein, 1998), phytoalexins in cotton (Zeringue, 1992), and systemin precursor in tomato (Sivasankar et al., 2000). Furthermore, GLVs induce a number of oxylipin and phenylpropanoid biosynthetic genes in Arabidopsis (Bate and Rothstein, 1998), bean (Arimura et al., 2000), and citrus (Gomi et al., 2003). C6-volatiles have been suggested to be essential in the activation of wound-related pathways, some of which are JA independent (Bate and Rothstein, 1998). This notion was supported by the analysis of downstream effects of separately silencing AOS and HPL branches of the 13-LOX pathway (Duan et al., 2005). Existing data also demonstrate that AOS- and HPL-derived signalling compounds execute co-operative defence signalling via molecular cross-talk (Halitchke et al., 2004). To date, the function of oxylipins derived from other 13-LOX-dependent enzymatic pathways is still poorly understood.
Despite some progress made in the functional analysis of LOXs in diverse dicot species, very little is known about the physiological significance of these enzymes in monocots. Several members of the LOX gene families were cloned or analysed at the protein level from major crop monocot species such as barley, wheat, and rice (Peng et al., 1994; Bohland et al., 1997; Mauch et al., 1997; Vörös et al., 1998; Mizuno et al., 2003; Agrawal et al., 2004). In the only reported functional analysis of a monocot LOX, overexpression of the rice 13-LOX gene RCI-1 resulted in increased levels of pathogenesis-related protein PR-1 transcripts (Zabbai et al., 2004). This study and other studies conducted on barley (Hause et al., 1999; Weichert et al., 1999) suggested that 13-LOXs may be involved in the activation of acquired resistance.
The genome of maize harbours at least 12 LOX genes (Kolomiets et al., 2004). Only two of these genes have undergone molecular and biochemical characterization (Wilson et al., 2001; Kim et al., 2003). cssap92 (designated as ZmLOX3 in Fig. 2), a predominantly 9-LOX, is upregulated during seed germination and infection with Aspergillus flavus and Fusarium verticillioides (Wilson et al., 2001). The other published maize LOX gene is reportedly a mixed function LOX that possesses both 9- and 13-LOX activity (Kim et al., 2003) and is a member of the type 1-LOX subfamily (designated ZmLOX1 in Fig. 2). As a first step in achieving the long-term goal of better understanding the function of 13-LOXs and their metabolites in maize interactions with mycotoxigenic fungi, here the isolation and molecular and biochemical characterization of two novel chloroplast-targeted 13-LOX genes, designated ZmLOX10 and ZmLOX11, are reported.
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Materials and methods |
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Isolation of genomic DNA and Southern blot analysis
Freshly harvested leaves from 2-week-old maize (Zea mays L.) seedlings were used for extraction of genomic DNA as described (Zhang et al., 2005). For Southern blot assay, genomic DNA (10 µg) from several different inbred lines was cut with various restriction enzymes: EcoRI, BamHI, HindIII, XhoI, and XbaI. Digested DNA was separated on a 0.8% agarose electrophoresis gel, transferred (0.025 M phosphate transfer buffer), and then cross-linked to the nylon membrane (Magna Nylon Transfer Membrane, Osmonics Inc., Minnetonka, MN, USA) by UV StratalinkerTM 2400. The blots were hybridized overnight at 42 °C with the 32P-labelled ZmLOX10-specific probe. The probe was 428 bp long and consisted of the 3' portion of the gene including 402 bp before and 24 bp after the stop codon. The membrane was hybridized in ULTRAhyb hybridization buffer (Ambion, Austin, TX, USA). Washes were performed: first with low stringency buffer (2x SSC and 0.1% SDS) at 42 °C twice for 5 min, followed by an additional wash with high stringency buffer (0.2 M SSC and 0.1% SDS) twice at 42 °C for 15 min. The blots were exposed to X-ray film (Kodak, Rochester, NY, USA) in cassettes at –80 °C for 72 h unless otherwise indicated.
Plant material and treatments
Maize plants were grown in a growth chamber in 7 cm pots in Strong-Lite® potting soil (Universal Mix, Pine Bluff, AZ, USA), temperature was maintained from 22–30 °C under a 16 h daylength, 50% average relative humidity, 560–620 µE of light. For all of the organ-specific expression studies (except for the ear, the tassel, and the silk tissues), wounding, and hormonal treatments 2-week-old maize seedlings of inbred line B73 at the V3 developmental stage were used. Tassel tissue was harvested at the time of pollen shedding, ears at the stage of silk emergence, and unpollinated silks were harvested from B73 plants grown in the field, and used for total RNA extraction and mRNA blot analyses. For wounding experiments, the second fully expanded leaves at the V3 stage were wounded by crushing the lamina with the haemostat. Cold treatment of plants at the V2 stage was conducted by shifting the seedlings to 4 °C for 24 h, whereas control plants were kept at room temperature (22 °C). For hormonal treatments, the seedlings were cut at the soil level and were incubated with the cut end placed in 100 ml of 0.01% Tween-20 water solution of either 200 µM MeJA, 2.5 mM salicylic acid (SA), or 100 µM abscisic acid (ABA) (Sigma, St Louis, MO, USA). As a control for this experiment, untreated plants were cut at the soil level and incubated in 0.01% Tween-20. Treatment with 10 µl l–1 ethylene was conducted in hermetically sealed 5.6 l desiccators. Entire ethylene-treated seedlings (two seedlings per replicate, three replicates) except for roots and mesocotyls were harvested at different time points after treatment, frozen immediately in liquid N2, and stored at –80 °C. For studies of circadian rhythm-regulated expression, seedlings of the B73 line were initially grown in a growth chamber at 22 °C with a 12 h photoperiod of 36 000–37 000 lux with the lights turned on at 08.00 h. When the seedlings reached the V2 stage they were divided into three groups: the first group was maintained at a 12 h/12 h light/dark photoperiod; the second group was transferred to a growth chamber with constant light; and the third group was transferred to constant darkness beginning at 02.00 h. All treatments were carried out in separate growth chambers (Percival Scientific, Inc., Perry, IA, USA). During the next 48 h, the second fully expanded leaves from two plants in each light treatment were harvested every 4 h and immediately frozen for subsequent RNA extractions.
Infection of plants with Cochliobolus carbonum
To study the LOX gene expression in a single genetic background during either compatible or incompatible interaction, near-isogenic strains of C. carbonum race 1 were used that either did (Tox2+) or did not (Tox2–) produce its pathogenicity factor, HC-toxin (kindly provided by Dr Guri Johal at Purdue University). For inoculation with C. carbonum, 2-week-old seedlings of inbred line Pr were used which is susceptible to C. carbonum Tox2+ wild-type strain but resistant to the Tox2– mutant strain (Multani et al., 1998). Conidial suspensions containing 105 conidia ml–1 in 0.01% Tween-20 were prepared as described in Meeley et al. (1992). Control plants were inoculated with sterile water (mock inoculated). Plants were inoculated by spraying the leaves to imminent run-off with conidial suspension. Following inoculation, plants were immediately placed under a plastic cover and were incubated for selected times in the greenhouse. After each incubation period, infected leaves were collected and frozen immediately in liquid N2 and stored at –80 °C until used for RNA isolations.
RNA extraction and northern and dot blot analysis
Total RNA from maize tissues was extracted by using TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA) according to the manufacturer's protocol. After extraction, 10 µg of total RNA from each experiment was separated using electrophoresis in 1.5% (w/v) formaldehyde agarose gels in 1x MOPS buffer and then transferred onto a MagnaGraph nylon membrane (Micron Separations Inc., Westboro, MA, USA). To confirm equal loading of RNA samples into an agarose gel and uniform transfer onto a nylon membrane for all experiments (except the C. carbonum inoculation study), rRNA was stained with ethidium bromide and was visualized by UV light. For the C. carbonum inoculation study, equal loading of total RNA was visualized by hybridization to the probes derived from 18S rRNA or tubulin. Because ZmLOX10 and ZmLOX11 genes share extremely high sequence identity within their coding sequence, the gene-specific probes were amplified by polymerase chain reaction (PCR) from the 3'-untranslated region (UTR) of the cDNAs where they share only limited identity. PCR primers for amplification of gene-specific probes were: lox10utrF1, 5'-ATC CTC AGC ATG CAT TAG TCC A-3'; lox10utrR1, 5'-AGT CTC AAA CGT GCC TCT T-3'; lox11utrF1, 5'-ACC CGT CCG TCC TCT CC-3'; and lox11utrR3, 5'-ATC CTA ATG GTA ACT CAA A-3'. The ZmLOX10 template was the SalI/NotI insert of the Pioneer p0006.cbysd84 expressed sequence tag (EST) clone. Genomic DNA extracted from the B73 inbred line was used as a template to PCR-amplify the probe for ZmLOX11, which then was sequenced to ensure its identity as the ZmLOX11 gene. To ensure the gene specificity of the probes, dot blot assay was performed by hybridization of the PCR fragments to denatured DNA of either the entire ZmLOX10 cDNA insert of p0006.cbysd84 plasmid or the genomic fragment of the 3'-UTR of ZmLOX11 generated by PCR as described above to the Hybond-N+ nylon membrane (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's protocols. Northern blot membranes were hybridized with 32P-labelled gene-specific probes and washed as described in Zhang et al. (2005). For autoradiography, RNA blots were exposed to a BioMax X-ray film (Kodak, Rochester, NY, USA) for 2–7 d at –80 °C, except for ZmLOX11 induction by ABA, where the exposure time was 14 d. Autoradiographic films were scanned and when necessary the relative intensity of the bands was determined using the publicly available Scion Image program (http://www.nist.gov/lispix/imlab/prelim/dnld.html). Relative quantification was done by comparing the intensities of ZmLOX10- and ZmLOX11-specific bands with corresponding RNA loading controls. Blots presented here are representative examples of at least two independent experiments.
Construction of overexpression vectors and analysis of the biochemical properties of ZmLOX10
The 5' end of the ZmLOX10 gene was cloned by the 5'-RACE (rapid amplification of cDNA ends) technique as described in Zhang et al. (2005). The entire open reading frame was PCR amplified using primers containing the BamHI site adopted for the in-frame start codon (5'-CGG-ATC-CAT-GAT-GAA-CCT-GAA-CCT-GAA-G-3') and the EcoRI site located after the stop codon (5'-CCT-TAA-GTC-AGA-TGG-ATA-TGC-TGT-GGG-G-3'). Following separation and elution from an agarose electrophoresis gel, the PCR fragment was inserted into the pCR2.1 TOPO® vector (Invitrogen, Carlsbad, CA, USA). After restriction digestion with BamHI/EcoRI, the coding region of ZmLOX10 was placed under the control of the isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible promoter of an Escherichia coli expression vector pET28a (EMD Biosciences, Inc., San Diego, CA, USA) using T4 DNA ligase (Roche Applied Science, Indianapolis, IN, USA). Following ligation, the pET28a expression construct containing ZmLOX10 was heat shock transformed into the BL21 (DE3) strain of E. coli (EMD Biosciences, Inc.). Two independent E. coli BL21 (DE3) cultures containing the expression constructs were grown overnight at 37 °C in LB medium (20 ml) containing 25 µg ml–1 kanamycin and 50 µg ml–1 ampicillin. When the cell culture reached a density value A600=0.7, 1 mM IPTG was added to induce expression of the constructs at 15 °C for 48 h. Cells collected from 200 ml of culture were resuspended in 30 ml of lysis buffer (50 mM TRIS–HCl, pH 7.5) that contained 10% (v/v) glycerol, 0.5 M NaCl, and 0.1% Tween-20. Cells then were disrupted by a sonifier tip with a frequency of five pulses per 30 s. In order to remove debris of cellular compartments, samples were centrifuged (12 000 g for 15 min). The supernatant was divided into 1 ml aliquots and stored at –20 °C (Feussner et al., 1998). Oxygenation of linoleic acid was carried out by incubating crude extract of bacteria containing the ZmLOX10 overexpression construct with the substrate (120 µM final concentration) diluted in 1 ml of 0.1 M sodium phosphate buffer (pH 5.7–7.0) or in 0.1 M Tris buffer (pH 7.7–8.6), for 20 min at room temperature. The reaction was stopped by the addition of 100 µl of glacial acetic acid. After centrifugation, the organic phase was vacuum dried to remove solvents. The remaining lipids were resuspended in 0.1 ml of high-performance liquid chromatography (HPLC) solvent. Detection of LOX products by HPLC was carried out on an Agilent (Waldbronn, Germany) 1100 HPLC system. The absorbances at 234 and 210 nm were recorded simultaneously. The enantiomer composition of the hydroxyl fatty acids was analysed as described by Feussner and Kühn (1995).
Bioinformatics analysis
A homology search using ZmLOX10 and ZmLOX11 translated sequences was done using the BLAST search which is publicly available at the National Center for Biotechnology Information (NCBI) web site (www.ncbi.nlm.nih.gov/BLAST/). The percentage identity of ZmLOX10 and ZmLOX11 to other plant LOXs was evaluated using ClustalW software available at the European Molecular Biology Laboratory, the European Bioinformatics Institute (EMBL-EBI) (www.ebi.ac.uk/clustalw/). For phylogenetic analysis, the deduced protein sequences of ZmLOX10 and ZmLOX11 were aligned with the sequences of published maize LOXs or plant LOXs whose physiological function was established by either antisense suppression or transgenic overexpression. For all the bioinformatics analyses, the default settings were used, unless indicated otherwise. The sequences were aligned using the Muscle program (Edgar, 2004). A phylogeny was reconstructed using the Neighbor–Joining method implemented in the Phylip package using Kimura's correction for multiple substitutions. The maximum likelihood tree was constructed using the parameters as described in Felsenstein (1989). Subcellular localization was predicted based on the identification of signal peptide sequences by four different programs ProtComp (www.softberry.com/berry.phtml), PSORT (psort.nibb.ac.jp/), TargetP (www.cbs.dtu.dk/services/TargetP/), and ChloroP (www.cbs.dtu.dk/services/ChloroP/). The presence of conserved domains in the deduced amino acid sequences was determined by NCBI Conserved Domain Search (www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and Pfam software (www.sanger.ac.uk/Software/Pfam/). Analysis of the ZmLOX10 promoter region for the presence of cis-acting elements and transcription factor-binding sites was done using the PLACE program (www.dna.affrc.go.jp/PLACE/) (Prestridge, 1991; Higo et al., 1999). To determine the genomic structure and identify promoter sequences of ZmLOX10 and ZmLOX11, a homology-based BLAST search was performed by utilizing the databases of the maize Genome Survey Sequences (GSS) (available at www.ncbi.nlm.nih.gov/BLAST/, www.plantgdb.org/PlantGDB-cgi/blast/PlantGDBblast, and http://tigrblast.tigr.org/tgi_maize/index.cgi) as well as Maize Assembled Genomic Island (MAGI) databases (available at www.plantgenomics.iastate.edu/maize/).
Distribution of materials
Novel materials described in this publication may be available for non-commercial research purposes upon acceptance and signing of a material transfer agreement. In some cases, such materials may contain or be derived from materials obtained from a third party. In such cases, distribution of material will be subject to the requisite permission from any third-party owners, licensors or controllers of all or parts of the material. Plant germplasm will not be made available except at the discretion of the owner and then only in accordance with all applicable governmental regulations. Obtaining any permission will be the sole responsibility of the requestor.
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Isolation and sequence analysis of ZmLOX10 and ZmLOX11 genes
To identify and clone maize 13-LOX genes potentially involved in biosynthesis of GLVs, the amino acid sequence from the GLV-producing potato 13-LOX gene (clone H1) (T07062 [GenBank] , Royo et al., 1996) was used in a BLAST search against the extensive DuPont/Pioneer and publicly available EST collections from Z. mays. This search identified 241 EST clones that belonged to a single contig and shared 57% identity at the deduced amino acid level with potato LOX H1. Sequence from the longest EST clone, named p0006.cbysd84, revealed that it represents only
2 kb of the 3' portion of the gene. Therefore, the 5' end of this gene was cloned by the 5'-RACE technique. The combined full-length cDNA sequence contained a complete open reading frame of 2718 bp and was named ZmLOX10 (GenBank accession no. DQ335768) in order to reflect its level of homology to ZmLOX1 as compared with the other 12 maize LOX genes identified (M Kolomiets, unpublished data). This sequence encodes a predicted 905 amino acid peptide with the estimated molecular mass of 102 kDa and an estimated pI of 6.57. The deduced amino acid sequence of ZmLOX10 exhibited the highest identity (72%) to the barley LOX2:Hv:3 gene (Q8GSM2; Bachmann et al., 2002) and 57% identity to tobacco NaLOX2 (AAP83137
[GenBank]
; Halitschke and Baldwin, 2003), both of which are type 2 13-LOXs.
Southern blot analysis was performed to determine the number of copies of the ZmLOX10 gene in the maize genome. The initial probe derived from ESTs consisted of 428 bp from the 3' portion of the gene, including 402 bp before and 24 bp after the stop codon. Under the most stringent conditions, for most of the five restriction enzymes and the three inbred lines, two bands of similar intensity were observed (Fig. 1), suggesting the existence of another closely related gene. To identify this missing gene, the newly cloned 5' portion of ZmLOX10 was used in a new BLAST search of the available EST collections. This search identified three EST clones that shared >95% nucleotide identity to the ZmLOX10 cDNA sequence. The complete sequence of the longest DuPont EST clone cds3f.pk006.d9a contained the 3' portion of the gene but lacked 215 amino acids of the N-terminus as predicted by comparison with ZmLOX10. This missing part of the gene was first identified by the BLAST search of available maize GSS (http://www.plantgdb.org/cgi-bin/PlantGDBblast) and was then cloned by 5'-RACE technique from cDNA derived from silks. Analysis of the combined cDNA sequence proved the presence of a new gene which shares 90% amino acid sequence identity to ZmLOX10 and was designated as ZmLOX11 (GenBank accession no. DQ335769). The full-length cDNA sequence for ZmLOX11 was 3125 bp long, encoding 911 amino acids with a predicted molecular mass of 103 kDa and estimated pI of 7.11. Despite their high sequence identity, ZmLOX10 and ZmLOX11 are discrete genes in the maize genome and were mapped to chromosomes 4 and 7, respectively, by using PCR with gene-specific primers and template genomic DNA from oat–maize chromosome addition lines (data not shown).
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Plant LOXs can be found in various cell organelles including chloroplast, microbodies, and cytoplasm (Feussner and Wasternack, 2002). In order to predict the subcellular localization of the LOX proteins, four different publicly available programs were used: ProtComp, PSORT, TargetP, and ChloroP. Analysis of ZmLOX10 and ZmLOX11 N-terminal sequences revealed the presence of a chloroplast targeting signal peptide with the cleavage site at positions 34 and 30, respectively (supplementary Fig. 1B available at JXB online). The closest homologue to both maize LOXs, the barley LOX2:Hv:3, was conclusively localized to chloroplasts (Bachmann et al., 2002). Supplementary Fig. 1A (available at JXB online) is an alignment of the predicted signal peptide sequences of ZmLOX10 and ZmLOX11 with the corresponding sequence of LOX2:Hv:3. The high degree of conservation provides strong support for the hypothesis that ZmLOX10 and ZmLOX11 are most probably localized to chloroplasts.
Similar to other known plant LOXs, the deduced amino acid sequences of both ZmLOX10 and ZmLOX11 contained residues required for iron binding and enzyme catalytic activity (Prigge et al., 1996): His559 and His564, His569 and His574, His754 and His760, Asn758 and Asn764, and Ile905 and Ile911, respectively (supplementary Fig. 1B available at JXB online). Moreover, it contains the Ser/Phe (621/622) conventional motif described to be indicative for plant 13-LOXs (Hornung et al., 1999). Analysis of the domain architecture performed using the Pfam program (supplementary Fig. 1C available at JXB online) showed that the proteins are comprised of two major domains: the lipoxygenase domain typical for all LOXs, and the PLAT domain known to mediate membrane attachment via other protein-binding partners (Marchler-Bauer et al., 2005).
Phylogenetic analysis
The phylogenetic relationship of ZmLOX10 and ZmLOX11 to other plant LOX functions which have been established were investigated to inform the predictions of biochemical and biological functions of these maize LOX genes. The maximum likelihood analysis identified two well-supported lineages of plant LOX proteins that appear to reflect rather accurately their regio-specificity and subcellular localization, cytoplasm-localized 9-LOXs and chloroplast-localized 13-LOXs (Fig. 2). ZmLOX10 and ZmLOX11 group together, with very strong bootstrap support, with chloroplast-localized 13-LOXs from both monocot and dicot species suggesting that these maize isoforms are most probably 13-LOXs. Within this group, the monocot proteins, barley LOX2:Hv:3 and rice RCI-1, group together with ZmLOX10 and ZmLOX11, suggesting that they are orthologues and thus may have a similar, as yet unidentified, function. Several members of the 13-LOX group such as Arabidopsis LOX2 and tobacco LOX3 are known to provide linolenic acid hydroperoxide substrates for the synthesis of JA in vivo (Bell et al., 1995; Halitschke and Baldwin, 2003) whereas others such as potato H1 and tomato LOXC are involved in the biosynthesis of C6-volatiles (Leon et al., 2002; Chen et al., 2004). Proteins with either predominant C9-positional specificity such as ZmLOX3 (Wilson et al., 2001) or a mixed C13/9 regio-specificity such as ZmLOX1 (Kim et al., 20003) clustered together with reasonable bootstrap support and are more distantly related to the group of 13-LOXs. Another maize LOX, ZmLOX12, encodes a 9-LOX (A Nemchenko et al., unpublished data) and was equally distant from either of the two major groups.
Genomic structure and predicted promoter cis-acting elements
Using the maize GSS and MAGI databases, overlapping genomic sequences covering the entire full-length cDNA of ZmLOX10 and ZmLOX11 and most of the promoter region for ZmLOX10 were identified. Genomic sequences with 98% or higher identity to the corresponding cDNA were exclusively selected to assemble genomic contigs for each of the two genes. The estimated length of genomic sequences (from the start to the stop codon) for ZmLOX10 (DQ459365
[GenBank]
) and ZmLOX11 (DQ459366
[GenBank]
) was 3360 and 3271 bp, respectively. Alignment of the cDNA with the corresponding genomic sequences showed overall similarity in the position, length, and composition of exons. Both genes are comprised of four exons and three introns. Differences between ZmLOX10 and ZmLOX11, however, were found in the length of the introns, with the first intron of ZmLOX10 being 4.3-fold longer than the first intron of ZmLOX11 (481 and 112 bp, respectively). However, the other two introns of ZmLOX11 were at least 1.5-fold longer than the corresponding introns of ZmLOX10. In both genes, the exon–intron–exon boundaries followed the rule of consensus junction sequences GT/AG (Mount, 1982).
Available GSS genomic sequences allowed the reconstruction of, presumably, the entire promoter region of the ZmLOX10 gene containing 1869 bp upstream of the start codon. By using the PLACE Signal Scan Search software (http://www.dna.affrc.go.jp/PLACE/; Higo et al., 1999), multiple putative cis-acting elements were detected in the ZmLOX10 promoter. Since the promoter sequence for ZmLOX11 was not available, it was not possible to compare it with the promoter of ZmLOX10. Supplementary Table 1 (available at JXB online) shows the categories of the regulatory sequence elements present in the promoter of ZmLOX10. These data were used as a guideline in further expression studies.
Biochemical properties of recombinant ZmLOX10
For further characterization of enzymatic properties, the ZmLOX10 coding region was amplified by PCR (described in the Materials and methods) and was placed under the control of an IPTG-inducible promoter of an E. coli expression vector. Crude extracts of bacteria containing the ZmLOX10 overexpression construct exhibited LOX activity. ZmLOX10 was most active at neutral to slightly alkaline pH conditions, with maximal LOX activity observed at pH 8.0 (data not shown). Therefore, regio-specificity was also determined at pH 8.0 using linoleic acid as a substrate. Similar results with regard to regio-specificity were regularly found when either linoleic or 
-linolenic acid was used as a substrate (Schaffrath et al., 2000). Since 96% of ZmLOX10-catalysed products were 13S hydroperoxides of linoleic acid, it was concluded that ZmLOX10 possessed clear 13-LOX regio-specificity. The preferred products of the ZmLOX10 enzymatic reaction detected by HPLC were 13-H(P)ODE [(9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid] and 13-KODE [(9Z,11E)-13-oxo-octadeca-9,11-dienoic acid] (Fig. 3). The fact that the hydro(pero)xides were predominantly (99.5%) S-enantiomers suggests their enzymatic origin rather than being a result of autocatalysis (Mueller et al., 2006). Amongst 13-LOX products, 13-H(P)ODE was preferentially produced (70%) as compared with 13-KODE (30%). The negative control did not show any detectable LOX activity.
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Organ-specific and circadian clock-regulated expression of ZmLOX10 and ZmLOX11
To rationalize the in vivo function of ZmLOX10 and ZmLOX11, expression of both genes was analysed by the northern blot technique. Gene-specific probes were generated based on 3'-UTR portions and tested by a dot blot assay. No cross-hybridization of the two probes to each other or to their corresponding full-length cDNAs was observed (Fig. 4A). Analysis of organ- and tissue-specific expression showed that the transcript levels of ZmLOX10 were most abundant in both young (V2 developmental stage) and older (R1 stage) leaves (Fig. 4B). ZmLOX10 is also expressed at relatively low levels in all rapidly growing tissues studied including developing tassel, silks, and shoot apical meristem. In contrast to ZmLOX10, expression of ZmLOX11 was the highest in silks and silk-bearing organs, ears. Moreover, expression of ZmLOX11 in silks was much higher than that of ZmLOX10, while its transcripts were at just detectable levels in tassel and shoot apical meristem. Such a differential distribution of transcripts for these two highly related genes in diverse organs suggests their dissimilar role during plant and organ development.
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Because the promoter region of ZmLOX10 harbours light-responsive cis-acting elements such as an I-box (see supplementary Table 1 at JXB online), it was hypothesized that expression of ZmLOX10 and/or ZmLOX11 may be regulated by light/dark conditions or the circadian clock. In order to test this hypothesis, V2 stage seedlings were subjected to different light treatments and leaf tissues were harvested at 4 h intervals over 2 d. Samples harvested within each treatment were analysed for steady-state mRNA levels of ZmLOX10 and ZmLOX11 using the northern blot assay (Fig. 5). Plants grown at a 12 h light/12 h dark photoperiod showed cyclic time-dependent fluctuations of transcript levels of ZmLOX10, with the maximum at 14.00 h (6 h after onset of light) and the minimum at 2.00 h (6 h after offset of light). The plants maintained the same pattern of expression for the rest of the duration of the experiment even after being transferred to constant light. When the plants were transferred into constant darkness, ZmLOX10 exhibited a similar cyclic expression pattern with a similar amplitude, but only during the first 24 h. However, no detectable fluctuation of ZmLOX10 transcripts was observed thereafter. This pattern is typically observed for other well-characterized plant circadian-regulated genes such as maize catalase 3 (Cat3; Polidoros and Scandalios, 1998). Figure 5 illustrates that accumulation of ZmLOX10 transcripts followed closely the pattern observed for Cat3 expression except that maximum ZmLOX10 expression preceded the highest levels of Cat3 transcripts by at least 4 h. This tendency was maintained throughout the time course of both light/dark and constant light experiments (Fig. 5A, B). However, in contrast to ZmLOX10, expression of Cat3 was notably reduced under conditions of constant dark. Closer examination of the available promoter region of ZmLOX10 revealed the presence of a promoter element, TTAATATCT, the so-called ‘site 2' (starts at a position 725 bp upstream from the start codon), that may confirm circadian rhythmicity for a number of Arabidopsis cycling genes (Harmer et al., 2000). ZmLOX11-specific mRNA was not detected throughout the entire time-course under all the dark/light conditions used in this experiment (Fig. 5).
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Wound-, cold-, and hormone-regulated expression of ZmLOX10 and ZmLOX11
The potential involvement of ZmLOX10 and ZmLOX11 in stress-induced defence responses was studied by examining mRNA accumulation levels in response to various defence-related hormones including JA, SA, ABA, and ethylene (Fig. 6A, B, D). As was the case with organ-specific expression, these genes were differentially expressed following most of the treatments applied. In leaves, exogenous JA transiently induced steady-state levels of ZmLOX10 transcripts starting at 3 h and reaching a maximum at 12 h after treatment (Fig. 6A). Similarly, wounding, known to induce endogenous accumulation of JA (Pena-Cortes et al., 1991), had the same effect on ZmLOX10 mRNA accumulation, with a maximum at 12 h (Fig. 6C). Available evidence suggests that JA and ethylene usually act synergistically in defence pathways, most probably via the involvement of the ERF1 transcription factor (Penninckx et al., 1996, 1998; Berrocal-Lobo et al., 2002; Lorenzo et al., 2003). In our study, however, mRNA levels of ZmLOX10 declined rapidly after treatment with ethylene and were minimal at 12 h post-treatment, but later started to increase and reached a maximum at 48 h (Fig. 6B). In control untreated plants, there was no secondary phase of mRNA accumulation detected (Fig. 6B). ZmLOX10 was also inducible by SA, with mRNA accumulation reaching the maximal levels at 6 h after treatment, but lower compared with those induced by JA. In sharp contrast to ZmLOX10, ZmLOX11 did not show any mRNA accumulation in response to JA, SA, ethylene, or wounding. However, transcript levels of both ZmLOX10 and ZmLOX11 were induced strongly by ABA as early as 6 h and 12 h post-application, respectively (Fig. 6D). Since, ABA was shown to be involved in cold stress responses, the accumulation of ZmLOX10 and ZmLOX11 in response to low temperature was measured. In this experiment, after exposing the young seedlings to chilling stress by shifting the plants from 22 °C to 4 °C, ZmLOX10 transcripts were induced rapidly and reached a maximum at 3 h, but declined thereafter (Fig. 7). However, no induction of ZmLOX11 transcripts was observed in response to chilling stress.
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ZmLOX10 but not ZmLOX11 is pathogen inducible
Induction of ZmLOX10 transcripts in response to JA and SA suggests that this gene may play an important role in maize defence reactions against pathogens. Therefore, it was elucidated whether expression of this gene can be altered differentially during compatible or incompatible plant–pathogen interactions. For this experiment, 2-week-old seedlings of the Pr inbred line were infected with two strains of a well-characterized necrotizing fungal agent of northern corn leaf spot C. carbonum race 1. Pr plants are susceptible to the HC-toxin-producing wild-type strain (Tox2+) and resistant to the mutant strain (Tox2–) that is deleted for HC-toxin production (Multani et al., 1998). Figure 8 illustrates that ZmLOX10 transcripts were induced to higher levels, reaching a maximum at 24 h post-inoculation during the incompatible interaction with the C. carbonum Tox2– strain. The compatible interaction with the HC-toxin-producing strain resulted in only a slight increase in mRNA accumulation comparable with the mock-inoculated controls. Most probably the low but detectable increases in the transcript levels in the mock- and Tox2+ strain-treated plants were due to fluctuations of gene expression driven by circadian rhythm. No induction of mRNA was observed for ZmLOX11 in response to infection by either pathogen tested (data not shown). These data suggest that ZmLOX10 but not ZmLOX11 may be specifically involved in defence mechanisms against necrotrophic fungal pathogens.
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Discussion |
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The biological significance of oxylipin metabolism initiated by 13-LOXs in any monocot species is little understood. To gain more insights into the role of maize 13-LOXs and their metabolites, in this study the cloning and characterization of two novel maize LOXs, ZmLOX10 and ZmLOX11, are reported. A high degree of identity between these two genes at both the cDNA (93%) and protein (90%) sequence levels, reflected by their extremely close clustering on the phylogenetic tree and their localization to different chromosomes, suggests that these genes evolved as a result of a relatively recent segmental duplication event. Both predicted proteins have a recognizable N-terminal chloroplast localization signal. In fact, ZmLOX10 is indeed localized to chloroplasts, as has been shown by a recent comparative proteomics study of maize bundle sheath and mesophyll chloroplasts (Majeran et al., 2005). This study described two LOX proteins that predominantly accumulated in the chloroplasts of mesophyll cells. One of these proteins (TC298873, formerly TC234252 in Majeran et al., 2005), is 100% identical to ZmLOX10 at the amino acid level. The recombinant ZmLOX10 is clearly a 13-LOX. On the grounds of their extremely high sequence homology, it is likely that ZmLOX11 possesses the same regio-specificity as ZmLOX10. Similarly to the present case, Tsitsigiannis et al. (2005) reported that another highly similar pair of 13-LOXs from peanut, PnLOX2 and PnLOX3, has identical biochemical properties, as is the case for the three plastidic 13-LOXs from barley (Vörös et al., 1998; Bachmann et al., 2002). Taken together, these data indicated that both predicted proteins are type 2 13-LOXs.
To understand the physiological function of ZmLOX10 and ZmLOX11, it is important to address the key question of what are the final oxylipin products of the pathway(s) initiated by these LOX isoforms. The comparison of ZmLOX10 and ZmLOX11 with the well-studied LOXs from other plant species indicated that the barley LOX2:Hv:3 gene is the closest monocot relative. LOX2:Hv:3 is also expressed predominantly in leaves and is proposed to be involved in channelling of linolenic acid substrate into the HPL-mediated production of C6-volatiles (Bachmann et al., 2002). Another close monocot homologue of these genes, the rice RCI-1, does not to contribute to JA biosynthesis (Zabbai et al., 2004). Moreover, antisense suppression of tobacco NaLOX2 (Halitschke and Baldwin, 2003) and potato LOXH1 (Leon et al., 2002), the two most closely related dicot homologues of ZmLOX10 and ZmLOX11, were devoid of C6-volatiles rather than JA. Therefore, it is hypothesized that the most likely biochemical function of ZmLOX10 and ZmLOX11 is in the HPL-mediated production of C6-aldehydes and alcohols and not in the biosynthesis of JA. In support of this hypothesis, ZmLOX10 expression in leaves in response to JA and wounding increased and peaked at least 3 h and 6 h later compared with JA- and wound-induced expression of ZmOPR7 and ZmOPR8 (Zhang et al., 2005). ZmOPR7 and ZmOPR8 are the only JA-producing OPRs (Zhang et al., 2005). Since the transcripts of the genes coding for the JA-producing enzymes are usually consecutively induced in response to JA (Sasaki et al., 2001) and OPRs are three enzymatic steps downstream of 13-LOXs, it is unlikely that ZmLOX10 supplies 13-hydroperoxylinolenic acid for JA production. This role is probably confined to three other 13-LOXs found in maize, some of which are induced by JA at much earlier time points (M Kolomiets et al., unpublished data).
Analysis of organ- and tissue-specific mRNA accumulation showed that despite their extremely high level of sequence similarity, ZmLOX10 and ZmLOX11 are expressed differentially in diverse organs of unchallenged plants. While ZmLOX10 is expressed predominantly in leaves, transcripts of ZmLOX11 accumulated abundantly in silks only, suggesting that this gene is silk specific. In agreement with this finding, only ZmLOX10 protein but not ZmLOX11 accumulates to high levels in mesophyll chloroplasts (Majeran et al., 2005). The present study showed that ZmLOX10 transcripts increased transiently by wounding, chilling, and treatments with JA, SA, and ABA, while ZmLOX11 was only induced by ABA, thus supporting the hypothesis that the physiological function of ZmLOX11 may be different from that of ZmLOX10. ABA was demonstrated to be a key regulator of plant responses to osmotic stress such as those associated with drought or chilling (Zeevaart and Creelman, 1988). Induction of ZmLOX10 during chilling stress and ABA suggests its involvement in maize responses to chilling and possibly other osmotic stress.
SA is a key signalling molecule required in the development of systemic acquired resistance (SAR) (Ryals et al., 1995) and it also mediates defence reactions against biotrophic pathogens (Glazebrook, 2005). Conversely, JA inhibits SA-induced expression of SAR (Traw et al., 2003), and is a key component of resistance mechanisms against necrotrophic pathogens and insects (Dong, 1998). Induction of ZmLOX10 by both JA and SA may seem rather surprising since numerous published reports indicated that JA and SA act antagonistically in defence reactions (Pena-Cortes et al., 1993; Feys and Parker, 2000; Cipollini et al., 2004). However, more recent studies suggest a more complex interaction between SA- and JA-mediated regulation of gene expression (Salzman et al., 2005; Mur et al., 2006). Similarly to ZmLOX10, a barley chloroplast-targeted 13-LOX is induced by both methyl jasmonate and SA (Weichert et al., 1999). ZmLOX10 was induced only during incompatible interactions with the avirulent strain of C. carbonum. Interestingly, accumulation of the highest levels of ZmLOX10 transcripts preceded the visual appearance of hypersensitive response (HR)-like necrotic lesions, suggesting its involvement in the HR-associated cell death. Similarly to ZmLOX10, a 13-LOX homologue from rice is induced only during the incompatible interaction of rice with rice blast fungus Magnaporthe grisea (Peng et al., 1994). Expression of ZmLOX11 was not induced by C. carbonum, thus suggesting no role for this duplicated gene in defences against this pathogen. Because of the observed strong induction of ZmLOX10 by JA, wounding, and pathogen infection, it is hypothesized that this gene and its products are primarily involved in defence responses to insects and pathogens.
To the best of our knowledge, this study is the first reported case of a plant LOX gene regulated transcriptionally by the circadian clock. The period of the increased accumulation of ZmLOX10 transcripts (10.00–18.00 h) coincided with the period of most active photosynthesis. Interestingly, a similar rhythm has been described for Arabidopsis genes encoding seven photosystem I and three photosystem II proteins that also peaked at around midday (Harmer et al., 2000). Additionally, the present data showed that the enzymatic activity of ZmLOX10 is the highest at pH 8.0, which is also within the range of physiological pH values during photosynthesis (Schaffrath et al., 2000). Based on the current surprisingly limited knowledge of diurnal regulation of lipid metabolism in plants, two alternative hypotheses are proposed to explain the possible physiological significance of clock-controlled ZmLOX10 expression. First, ZmLOX10 may fulfil a role proposed for 13-LOXs in the complex membrane lipid peroxidation during photosynthesis (Feussner et al., 1998). It is interesting that two circadian-regulated rat LOXs, 12-LOX and 5-LOX, also displayed their maximum activity during the day time and have been suggested to play key roles in the generation of reactive oxygen species, cell death, and calcium intracellular release (Reynaud et al., 1994; Li et al., 1997). The second hypothesis is that ZmLOX0 may be involved in the production of oxylipins with signalling activities, which in turn may help maize plants to adjust better to low night temperatures and, therefore contribute to chilling tolerance. Recent study of genome-wide expression of circadian-regulated Arabidopsis genes revealed that several enzymes involved in lipid modification were under the clock control and were proposed to have a role in the plant's adaptation to chilling temperatures during the night (Harmer et al., 2000). The fact that ZmLOX10 was induced early following the chilling treatment indirectly points towards this hypothesis. Coincidentally, the amounts of the LOX substrates linoleic and linolenic acid fluctuate in a light-dependent manner and are increased upon low temperature treatment (Rikin et al., 1993). Regardless of which of the proposed hypotheses is correct, diurnal regulation of ZmLOX10 is intriguing and warrants further examination.
In summary, based on mapping of ZmLOX10 and ZmLOX11 to different chromosomes and on extremely high sequence identity to each other, it is likely that this gene pair arose from a relatively recent segmental gene duplication event. A recent comparative study of two types of gene duplications, tandem (linked) and segmental (unlinked) duplications, in Arabidopsis suggested that (i) the average age of segmentally duplicated pairs is twice as high as that of tandemly duplicated genes; and (ii) perhaps because of this difference in time of gene divergence, a significantly larger proportion of segmentally duplicated genes shows a less similar expression pattern compared with tandemly duplicated genes (Haberer et al., 2004). A remarkably dissimilar expression pattern of ZmLOX10 and ZmLOX11 in all except ABA treatments fits this observation. This study represents an example of evolutionary employment of duplicated genes that seem to have distinct physiological functions despite their high overall homology. In order to determine conclusively the physiological function of the pathogen-inducible 13-LOX, ZmLOX10, Mu-transposable element insertional mutants for this gene were identified. Currently, we are in the process of introgressing these mutant alleles into the appropriate genetic backgrounds differing in their levels of resistance to diverse pathogens and pests.
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Supplementary data |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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We thank Dr Carl Simmons and Pedro Navarro (both at Pioneer Hi-Bred Intl) for their help with sequence analysis of EST clones representing the ZmLOX10 and ZmLOX11 genes. Dr Michael Thon (Texas A&M University) is acknowledged for his help with the phylogenetic analysis of plant LOXs. We thank Dr Heather Wilkinson (Texas A&M University) for critically reading the manuscript. We are grateful to Dr Deborah-Bell Pedersen for helpful advice on the circadian clock experiment. This research was supported in part by the Texas Corn Producers Board and by Texas A&M University and Texas Agricultural Experiment Station.
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Abbreviations |
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ABA, abscisic acid; AOS, allene oxide synthase; EST, expressed sequence tag; GLV, green leafy volatile; HPL, hydroperoxide lyase; IPTG, isopropyl-ß-D-thiogalactopyranoside; JA, jasmonic acid; LOX, lipoxygenase; OPR, 12-oxo-phytodienoic acid reductase; 5'-RACE, 5' rapid amplification of cDNA ends; SA, salicylic acid; SAR, systemic acquired resistance; UTR, untranslated region.
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References |
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