Soybean Receptor-like Protein Kinase Genes: Paralogous Divergence of a Gene Family

Etsuo Yamamoto and Halina T. Knap

Molecular Cytogenetics Laboratory, Clemson University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Receptor-like protein kinases (RLKs) in plants play major roles in cellular processes and stress responses. Three soybean (Glycine max) orthologs of Arabidopsis thaliana RLK were isolated and designated GmRLK1, GmRLK2, and GmRLK3. GmRLK1, GmRLK2, and GmRLK3 are similar in sequence, with GmRLK2 and GmRLK3 being nearly identical. The deduced amino acid sequences of GmRLK1, GmRLK2, and GmRLK3 possess characteristics of a transmembrane leucine-rich repeat RLK, AtCLV1. DNA fingerprinting and PCR analyses of a bacterial artificial chromosome library identified five GmRLK contigs (I–V): three for GmRLK1 (I, II, and V), one for GmRLK2 (III), and one for both GmRLK2 and GmRLK3 (IV). Phylogenetic analysis of the soybean RLKs together with other plant RLKs indicates that soybean and A. thaliana CLV1s generate a CLV1 branch, while soybean, A. thaliana, and rice RLKs generate an RLK branch. Thus, the AtCLV1 orthologs may have evolved later than the other pathogen–, environmental stress–, plant hormone–, and development–associated RLKs. A common ancestral GmRLK gene may have duplicated to give rise to GmRLK1, GmRLK2, and GmRLK3, or GmRLK2 and GmRLK3 may have resulted from a recent duplication event(s). Several amino acid replacements in the kinase domain of GmRLK1 compared with those of GmRLK2 and GmRLK3 may reflect evolutionary divergence of individual family members.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Receptor protein kinases comprise multigene families. They are plasma membrane–bound and play an important role in the perception and transmittance of external signals (Fantl, Johnson, and Williams 1993Citation ; Braun and Walker 1996Citation ). Many signals are perceived by the extracellular domains of receptor protein kinases and are transduced by activation of intracellular kinase domains. Receptor protein kinases in animals can autophosphorylate either on tyrosine residues or on serine and/or threonine residues. Plant counterparts, receptor-like protein kinases (RLKs), have topological features of the tyrosine receptor protein kinases but contain sequence motifs characteristic of serine/threonine kinases. Plant RLK gene families participate in wide spectra of cellular responses, and functional constraints on their DNA sequences make them excellent subjects to trace gene genealogy at the within-species level.

Plant RLKs are classified into three major groups (Braun and Walker 1996Citation ). The first group includes RLKs with an extracellular S-domain, characteristic of the S-locus receptor kinases (SRKs) and the S-locus glycoproteins (SLGs) from Brassica (Nasrallah and Nasrallah 1993Citation ). SRKs and SLGs are expressed in the stigma and have been implicated in inhibition of self-pollination (Nasrallah and Nasrallah 1993Citation ; Takasaki et al. 2000Citation ).

The second group includes RLKs with novel extracellular domains. A unique extracellular domain was identified in Catharanthus roseus CrRLK1 (Schulze-Muth et al. 1996Citation ). In contrast to other plant RLKs, CrRLK1 uses an intramolecular rather than an intermolecular phosphorylation mechanism (Schulze-Muth et al. 1996Citation ). In Arabidopsis thaliana RLK Wak1, an extracellular domain with several epidermal growth factor repeats and a region similar to a viral movement protein was identified (He, Fujiki, and Kohorn 1996Citation ). Wak1 is expressed in vegetative tissues, and its expression is induced by pathogen and salicylic acid (He, He, and Kohorn 1998Citation ).

The third group of RLKs belongs to the leucine-rich repeat (LRR) protein superfamily (Kobe and Deisenhofer 1994Citation ; Jiang et al. 1995Citation ). Plant LRR-RLKs play major roles in cellular functions, including regulation of endosperm and pollen development (Li and Wurtzel 1998Citation ; Muschietti, Eyal, and McCormick 1998Citation ), regulation of meristem and flower development (Torii et al. 1996Citation ; Clark, Williams, and Meyerowitz 1997Citation ; Kim, Jeong, and An 2000Citation ), floral organ abscission (Jinn, Stone, and Walker 2000)Citation , gibberellin-induced stem elongation (van der Knaap et al. 1999Citation ), brassinosteroid signal transduction (Li and Chory 1997Citation ), environmental stress responses (Hong et al. 1997Citation ), and disease resistance (Song et al. 1995Citation ). Thus, plant LRR-RLKs respond to a wide range of extracellular signals and transduce intracellular responses depending on the nature of the signal and cellular localization.

In A. thaliana, a putative membrane-bound LRR-RLK, AtCLV1, is involved in shoot and floral meristem development (Clark, Running, and Meyerowitz 1993Citation ; Clark, Williams, and Meyerowitz 1997Citation ). A GenBank search identified several putative A. thaliana LRR-RLKs that were similar in sequence to AtCLV1. One of these LRR-RLKs was characterized. The deduced amino acid sequence of the AtRLK gene (GenBank accession number CAA16688) contains characteristics of a membrane-bound LRR-RLK: an N-terminal signal peptide, an extracellular LRR domain, a transmembrane region, and a cytoplasmic serine/threonine kinase domain (Braun and Walker 1996Citation ). The sequence identity of AtRLK and AtCLV1 is 48% in regions containing potential signal peptides, LRR domains, and transmembrane regions, while it is 81% in regions containing potential kinase domains.

We previously isolated and characterized two soybean (Glycine max) LRR-RLK genes, GmCLV1A and GmCLV1B (Yamamoto, Karakaya, and Knap 2000)Citation , homologs of AtCLV1. In this study, we isolated and characterized three other LRR-RLK family members, GmRLK1, GmRLK2, and GmRLK3, orthologs of AtRLK. A phylogenetic analysis of the deduced amino acid sequences of these soybean RLKs together with RLKs of several other plant species suggests that AtCLV1 orthologs may have developed later in the evolution of the plant RLK multigene families.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
RNA and DNA Isolation
Seven soybean genotypes, BARC-11-11-ff, Clark 63, Essex, Faribault, PI 209332, PI 437654, and Williams 82 were grown in the field and greenhouse. Leaves, vegetative-shoot apices, reproductive-floral apices, and roots were excised from the plants and frozen. Poly(A)+RNA was isolated directly from the tissues using the FastTrack 2.0 mRNA isolation kit following the manufacturer's protocol (Invitrogen, Carlsbad, Calif.). Genomic DNA was extracted from leaves according to published procedures (Keim, Olson, and Shoemaker 1988Citation ).

Complementary DNA Isolation, Reverse Transcriptase–Polymerase Chain Reaction, and Rapid Amplification of cDNA Ends
cDNA was synthesized from mRNA by Maloney murine leukemia virus reverse transcriptase (RT). Polymerase chain reaction (PCR) was performed using degenerate forward (RLKF) and reverse (RLKR) primers (table 1 ) designed from the conserved kinase domains of AtRLK, AtCLV1, and Oryza sativa (GenBank accession numbers CAA16688, U96879, and X89226, respectively). PCR-amplified fragments were gel-purified, cloned into pCR2.1 (Invitrogen) or pGEM-T Easy (Promega, Madison, Wis.) vector, and sequenced with an ABI 373 automated sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems, Foster City, Calif.). The regions of the open reading frame (ORF) were resequenced by direct-sequencing of RT-PCR products. Rapid amplification of cDNA ends (RACE) was performed using the SMART RACE amplification kit (Clontech, Palo Alto, Calif.). Gene-specific primers for 3' RACE were IF3', IIF3', and IIIF3' (table 1 ), and gene-specific primers for 5' RACE were IR5', IIR5', and IIIR5' (table 1 ).


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Table 1 PCR Primers for cDNA Isolation and Genomic Analyses of GmRLK1, GmRLK2, and GmRLK3 in Soybean

 
Sequence Analyses and Algorithms
Sequence analyses were performed using BCM Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/), ENTREZ/BLAST (http://www.ncbi.nlm.nih.gov/), ExPASy (http://www.expasy.ch/), TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html), ALIGN (http://vega.crbm.cnrs-mop.fr/bin/align-guess.cgi), PSITE (http://dot.imgen.bcm.tmc.edu:9331/pssprediction/pssp.html), PSORT (http://psort.nibb.ac.jp), and SOSUI (http://azusa.proteome.bio.tuat.ac.jp/sosui/).

DNA Blot Analysis
Genomic DNA (10 µg) from genotypes BARC-11-11-ff and Clark 63 was digested with XbaI and EcoRV and loaded on a 0.8% agarose gel. The gel was depurinated, denatured, and blotted onto a Hybond N+ membrane (Amersham, Piscataway, N.J.). The membrane was prehybridized in a solution of 6 x SSC, 5 x Denhardt's reagent, 0.5% SDS, and 100 µg/ml salmon sperm DNA at 65°C for 2 h and hybridized with a probe derived from the 3' untranslated regions (UTRs) of GmRLK1 (IF-IR, 262 bp), GmRLK2 (IIF-IIR, 215 bp), or GmRLK3 (IIIF'-IIIR', 193 bp) (table 1 ) at 65°C for 16 h. PCR conditions were as follows: denaturation at 95°C for 1 min; five cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s; 25 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and extension at 72°C for 3 min. PCR products were gel-purified and resuspended in 50 µl of H2O. Two milliliters of the resuspended DNA was used for PCR labeling with 50 µCi 32P dCTP in a 20-µl reaction volume. PCR conditions were the same as previously stated. The membrane was washed in 2 x, twice in 1 x, and once in 0.1 x SSC containing 0.1% SDS at 65°C for 45 min and exposed to X-ray film at -70°C.

Screening of Soybean BAC Clones by DNA Fingerprinting and PCR
A soybean BAC library that covers nine genome equivalents (Tomkins et al. 1999Citation ) was screened independently with GmRLK1, GmRLK2, and GmRLK3 probes. BAC clones in the microtiter plates were identified from the autoradiograph. BAC DNA was isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.). For BAC DNA fingerprinting, DNA was digested with HindIII, fractionated on a 1% agarose gel, and analyzed using FPC V4.6.1 (Sanger Center, Cambridge, U.K.). Parameters for contig assignment were a tolerance value seven and cutoff scores of 10-4–10-12. The presence of GmRLK1, GmRLK2, and GmRLK3 in the BAC clones was examined by PCR using gene-specific primers. Gene-specific primers were GmRLK1 (IF and IR), GmRLK2 (IIF and IIR), and GmRLK3 (IIIF and IIIR) (table 1 ) or GmCLV1A (1A3'5F and 1A3'6R) and GmCLV1B (1B3'7F and 1B3'8R) (Yamamoto, Karakaya, and Knap 2000)Citation . PCR conditions for primary screening were as follows: denaturation at 95°C for 1 min and 30 s; two cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s; three cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s; 25 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and extension at 72°C for 3 min. PCR conditions for secondary screening were as follows: denaturation at 95°C for 1 min and 30 s; 20 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s; and extension at 72°C for 3 min. PCR conditions for tertiary screening were as follows: denaturation at 95°C for 1 min and 30 s; two cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s; 22 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and extension at 72°C for 3 min. PCR products were fractionated on a 1% agarose gel.

Relative Multiplex Quantitative RT-PCR
Expression of GmRLK1, GmRLK2, and GmRLK3 was examined by relative multiplex quantitative RT-PCR using the soybean proteasomal IOTA subunit gene as the internal control (Boissonneault and Lau 1993Citation ; Spencer and Christensen 1999Citation ; Yamamoto, Karakaya, and Knap 2000Citation ). The soybean IOTA subunit gene is expressed constitutively and ubiquitously in soybean (Tang 1999Citation ). The IOTA fragments (303 bp) were amplified using IO5' and IO3' primers (Tang 1999Citation ), and GmRLK fragments were amplified using gene-specific GmRLK1 (IF-IR), GmRLK2 (IIF-IIR), GmRLK3 (IIIF'-IIIR'), and GmRLK4 (IVF-IVR) primers (table 1 ). GmRLK4, a gene expressed only in root that was isolated at our laboratory (unpublished data), was included to show that there was no DNA contamination in the starting mRNA. GmRLK4 primers amplify 382-bp fragments. PCR conditions were as follows: denaturation at 95°C for 1 min; one cycle of 95°C for 30 s, 66°C for 30 s, and 72°C for 40 s; two cycles of 95°C for 30 s, 64°C for 30 s, and 72°C for 40 s; two cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 40 s; 20 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 40 s; and extension at 72°C for 3 min. PCR products were fractionated on a 4% (w/v) Nusieve 3:1 agarose gel (FMC, Rockland, Maine), stained with ethidium bromide, and photographed. The relative intensities of the bands in each lane were quantitated by scanning the gel with the fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan), and the areas of the peaks were compared with that of GmIOTA as the relative control.

Phylogenetic Analysis of Plant RLKs
Phylogenetic analysis of GmRLK1, GmRLK2, GmRLK3, GmCLV1A, GmCLV1B, AtRLK, and AtCLV1 (GenBank accession numbers AF244888, AF244889, AF244890, AAF59905, AAF59906, CAA16688, and AAB58929, respectively), together with other plant RLKs (A. thaliana AtHAESA, AtER, AtBR1, AtWak1, and AtRPK1, rice OsTMK and OsXa21, Brassica BoSRK3, maize ZmRLK2, tomato LePRK1 [GenBank accession numbers P47735, U47029, AF017056, AJ009696, U55875, Y07748, U72724, X79432, AF023165, and U58474, respectively], and rice OsLRK1 [Kim, Jeong, and An 2000]), was conducted with GeneBee (http://www.genebee.msu.ru/services/malign_full.html) using the Dayhoff amino acids distance matrix (Brodsky et al. 1993Citation ). Phylogenetic trees were generated by cluster and topological algorithms using GeneBee-Net v.2.0 (http://www.genebee.msu.ru/services/phtree_reduced.html), and the branch lengths were adjusted by the Fitch-Margoliash method with contemporary tips, version 3.572c (http://sdmc.krdl.org.sg:8080/~lxzhang/phylip/).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sequence Comparison of Soybean and Arabidopsis RLKs
Soybean cDNAs whose sequences are homologous to that of AtRLK (GenBank accession number CAA16688) were isolated. Three soybean homologs of AtRLK were designated GmRLK1 (3,350 bp), GmRLK2 (3,123 bp), and GmRLK3 (3,335 bp). The pairwise sequence identities between GmRLK1, GmRLK2, and GmRLK3 ranged from 80% to 95% in the ORFs (global alignment). The deduced amino acid sequences of AtRLK, GmRLK1, GmRLK2, and GmRLK3 are shown in figure 1 . The predicted Mr and pI were 109,203 and 6.0 for AtRLK; 109,725 and 6.5 for GmRLK1; 110,116 and 6.2 for GmRLK2; and 110,322 and 6.2 for GmRLK3. The pairwise sequence identities of the deduced amino acid (aa) sequences between GmRLK1 (1,008 aa), GmRLK2 (1,012 aa), and GmRLK3 (1,012 aa) ranged from 85% to 96% (global alignment). GmRLK2 and GmRLK3 were almost identical (fig. 1 ). The pairwise sequence identities between AtRLK (1,003 aa) and GmRLK1, GmRLK2, and GmRLK3 were 78%, 79%, and 79% (global alignment), respectively. The kinase domains were highly conserved (fig. 1 ). The sequence identities between GmCLV1A and GmCLV1B and GmRLK1, GmRLK2, and GmRLK3 ranged from 41% to 42% in regions containing signal peptides, LRRs, and transmembrane regions, while the sequence identities were 79%–81% in regions containing kinase domains.



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Fig. 1.—Sequence alignments of the deduced amino acids of Arabidopsis thaliana and soybean receptor-like protein kinase (RLK) genes. Sequence alignments were performed with MAP (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html) and viewed with BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Regions of identity (black), similarity (shaded), and differences (white) are indicated. °, paired cysteine residues; *, missense mutation site in A. thaliana CLAVATA1 (Clark, Williams, and Meyerowitz 1997Citation ); ·····, RT-PCR primer site in the kinase domain; ++++, predicted transmembrane region; I–XI, conserved kinase subdomains. The possible signal peptide cleavage sites are located between amino acid residues 16 and 17 for AtRLK, between residues 16 and 17 for GmRLK1, between residues 22 and 23 for GmRLK2, and between residues 15 and 16 for GmRLK3 (PSORT; http://psort.nibb.ac.jp). GenBank accession numbers are CAA16688 for AtRLK, AF244888 for GmRLK1, AF244889 for GmRLK2, and AF244890 for GmRLK3. The EMBL alignment number is DS42518

 
The N-termini of AtRLK, GmRLK1, GmRLK2, and GmRLK3 contain potential hydrophobic-signal peptides that may direct the secretion of these proteins to the plasma membrane (PSORT server; Stein et al. 1996Citation ). These putative signal peptides are followed by the putative extracellular regions consisting of conserved LRR domains (fig. 2 ). The LRR domains of AtRLK (amino acid residues 74–606), GmRLK1 (amino acid residues 67–598), GmRLK2 (amino acid residues 72–603), and GmRLK3 (amino acid residues 72–603) contain 22 complete LRRs (fig. 2 ) (AtRLK not shown). The LRR domains are flanked by pairs of conservatively spaced cysteine residues (fig. 1 ). The majority of the potential N-glycosylation sites (N-X-S/T-X) are located in the LRR domains (figs. 1 and 2 ). The predicted transmembrane regions reside at amino acid residues 642–661 in AtRLK, 634–653 in GmRLK1, 638–657 in GmRLK2, and 638–657 in GmRLK3 (fig. 1 ), and these peptides may function as type Ia signal anchors (PSORT server; High and Dobberstein 1992Citation ). The predicted transmembrane regions of AtRLK, GmRLK1, GmRLK2, and GmRLK3 are highly hydrophobic and are predicted to form {alpha}-helices in the membranes (PSORT and SOSUI servers). The transmembrane regions are followed by a positively charged cytoplasmic stop-transfer sequence (High and Dobberstein 1992Citation ; Schulze-Muth et al. 1996Citation ). The putative intracellular, cytoplasmic tail regions contain all of the conserved kinase subdomains found in serine/threonine protein kinases (fig. 1 ).



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Fig. 2.—The leucine-rich repeat (LRR) domains of soybean receptor-like protein kinases (RLKs). A, LRR domain of GmRLK1 (amino acid residues 67–598). B, LRR domain of GmRLK2 (amino acid residues 72–603). C, LRR domain of GmRLK3 (amino acid residues 72–603). The LRR domains are aligned showing 22 complete repeats. {Phi}, hydrophobic amino acid; –, any amino acid; =, frequently D or E; {equiv}, S, T, or {Phi}. E, ß-sheet; L, loop; H, {alpha}-helical. Aligned by the methods of Jiang et al. (1995)Citation and Clark, Williams, and Meyerowitz (1997)Citation

 
Several amino acid replacements are found in GmRLK1, GmRLK2, and GmRLK3. In GmRLK1, Thr-842 and Pro-919 are replaced by Ala and Ser, respectively, in the kinase domain compared with those of AtRLK, GmRLK2, and GmRLK3 (fig. 1 ). At the conserved ß-casein phosphorylation site, His-828 in AtRLK, GmRLK2, and GmRLK3 is replaced by Phe in GmRLK1 (fig. 1 ). Furthermore, in the LRR domains, GmRLK2 and GmRLK3 lack aminotransferase class-I-pyridoxal-phosphate attachment sites due to amino acid replacements Lys-402 to Thr in GmRLK2 and Lys-402 to Ser in GmRLK3, while GmRLK1 and AtRLK retain the sites (fig. 1 ). Amino acid replacements at the conserved kinase domain and the other domains between GmRLK1 and GmRLK2/GmRLK3 suggest that GmRLK2 and GmRLK3 have undergone a paralogous evolution.

Genomic Organization of the Soybean RLK Genes
DNA gel blot analysis was conducted to identify potential restriction fragment length polymorphisms (RFLPs) for GmRLK1, GmRLK2, and GmRLK3 using the gene-specific 3' UTR probes. No RFLPs were found between seven genotypes (two primitive genotypes, PI 209332 and PI 437654; five advanced cultivars, BARC-11-11-ff, Clark, Essex, Faribault, and Williams 82) with 10 restriction enzymes, implying that restriction sites are not rearranged within the genes or at the flanking regions of the GmRLK genes. The presence of one or two bands in each restriction digestion indicates that GmRLK1, GmRLK2, and GmRLK3 are transcribed from low-copy-number genes (restriction patterns of two genotypes digested with XbaI and EcoRV are shown in figure 3 ). The GmRLK1 probe detects one major band (fig. 3 , lanes 1, 2, 7, and 8). The GmRLK2 probe detects two restriction bands (fig. 3 , lanes 3, 4, 9, and 10). The GmRLK3 probe (IIIF' and IIIR', 193 bp; table 1 ) detects the lower-molecular-weight band of the GmRLK2 bands (fig. 3 , lanes 5, 6, 11, and 12). This result shows that the GmRLK2 and GmRLK3 genes, despite high sequence similarities in the ORFs (95%), could be distinguished by gene-specific primers which were designed from the divergent 3' UTRs of individual family members. Thus, the higher-molecular-weight band contains the GmRLK2 gene (fig. 3 , lanes 3, 4, 9, and 10), and the lower-molecular-weight band contains the GmRLK2 and GmRLK3 genes (fig. 3 , lanes 3–6 and 9–12).



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Fig. 3.—DNA gel blot analysis of soybean receptor-like protein kinase (RLK) genes. Genomic DNA (10 µg) was digested with XbaI and EcoRV. Lanes 1–6, XbaI; lanes 7–12, EcoRV. Lanes 1, 2, 7, and 8, GmRLK1; lanes 3, 4, 9, and 10, GmRLK2; lanes 5, 6, 11, and 12, GmRLK3. Lanes 1, 3, 5, 7, 9, and 11, genotype Clark 63; lanes 2, 4, 6, 8, 10, and 12, BARC-11-11-ff. The membrane was hybridized with a GmRLK1 (IF-IR, 262 bp), GmRLK2 (IIF-IIR, 215 bp), or GmRLK3 (IIIF'-IIIR', 193 bp) probe. Approximate molecular lengths of lambda HindIII markers are indicated at the right

 
In order to detect genomic regions containing GmRLK1, GmRLK2, and GmRLK3 in the soybean genome, a soybean BAC library (Tomkins et al. 1999Citation ) was screened with gene-specific GmRLK1, GmRLK2, and GmRLK3 probes independently (table 1 ). Forty BAC clones were obtained. The presence of GmRLK1, GmRLK2, and GmRLK3 in the 40 BAC clones was confirmed by PCR analysis using the gene-specific primers (table 1 ) (data not shown). BAC DNA fingerprinting was evaluated to identify possible genomic diversification at GmRLK loci. Five nonoverlapping contigs (I–V) were assembled. BACs in contigs I, II, and V were detected with a GmRLK1 probe, BACs in contig III with a GmRLK2 probe, and BACs in contig IV with both GmRLK2 and GmRLK3 probes. Thus, the soybean genome contains three regions for GmRLK1 and two regions for GmRLK2, implying that gene duplication was accompanied by chromosomal segmental rearrangements. One of the two GmRLK2 regions also contains GmRLK3, and these GmRLK genes are tandemly arranged. This result agrees with the DNA restriction patterns, showing that GmRLK2 and GmRLK3 are clustered in the lower-molecular-weight band (fig. 3 , lanes 3–6 and 9–12).

Expression Analyses of the GmRLK Genes
Expression analysis of GmRLK1, GmRLK2, and GmRLK3 was performed by relative multiplex quantitative RT-PCR using poly(A)+RNA as templates (Boissonneault and Lau 1993Citation ; Spencer and Christensen 1999Citation ; Yamamoto, Karakaya, and Knap 2000Citation ). GmRLK4, an RLK with root-specific expression, was included in the experiment. GmRLK4 is a cDNA fragment (571 bp) that shares sequence similarity with common bean RLK PvRK20-1 (51% identity in 137 aa) (Lange et al. 1999Citation ). PvRK20–1 is expressed in roots, and its expression is induced by pathogens and nodulin factors. Gene-specific GmRLK4 primers (table 1 ) amplify a 382-bp-long fragment, which preferentially accumulates in soybean roots (fig 4 , lane 6). No transcript detection with GmRLK4 primers in leaves and shoot apices (fig. 4 , lanes 2 and 3) indicates no DNA contamination in the mRNA. Visible amplification of GmRLK1, GmRLK2, and GmRLK3 transcripts in roots, leaves, and vegetative-shoot and reproductive-floral apices implies that the GmRLK1, GmRLK2, and GmRLK3 genes are expressed in all organs in soybean (fig. 4 ). The significant finding is that expression of GmRLK1 is most prominent in vegetative-shoot apices (fig. 4 , lanes 3 and 4). Expression of GmRLK1 in vegetative-shoot apices is 30%–40% more extensive than that of leaves, reproductive-floral apices, and roots (fig. 4 , lanes 2, 5, and 6). Despite the high sequence similarities of GmRLK2 and GmRLK3 (96% identity; fig. 1 ), there are small differences in the expression patterns of GmRLK2 and GmRLK3 (fig. 4 ).



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Fig. 4.—Expression analysis of soybean receptor-like protein kinase (RLK) genes by relative multiplex quantitative RT-PCR. Lane 1, 50-bp DNA ladder; lane 2, leaf; lanes 3 and 4, vegetative-shoot apex; lane 5, reproductive-floral apex; lane 6, root. Lanes 2, 3, 5, and 6, genotype Clark 63; lane 4, BARC-11-11-ff. GmRLK4 (IVF-IVR, 382 bp); GmIOTA (IO5'-IO3', 303 bp, internal control); GmRLK1 (IF-IR, 262 bp); GmRLK2 (IIF-IIR, 215 bp); GmRLK3 (IIIF'-IIIR', 193 bp). The amplified RT-PCR products are stained with ethidium bromide. Numbers indicate the relative amounts of DNA in the bands normalized with GmIOTA as the control (100)

 
Phylogenetic Analysis of Plant RLKs
In order to determine evolutionary lineages of the GmRLK gene family, a phylogenetic tree for soybean GmRLK1, GmRLK2, GmRLK3, GmCLV1A, GmCLV1B, AtRLK, and AtCLV1, together with several RLKs of other plant species, was constructed. The phylogenetic tree indicates that a common ancestral gene may have duplicated to give rise to CLV1 and RLK lineages before the split of soybean, A. thaliana, and rice (fig. 5 ). GmCLV1A and GmCLV1B cluster, and together with AtCLV1, they form a CLV1 branch. Three GmRLKs together with AtRLK and OsLRK1 form an RLK branch. The phylogenetic tree also indicates that these AtCLV1 orthologs, together with A. thaliana floral abscission AtHAESA, may have evolved later than the other developmental (AtER, BoSRK3, ZmRLK2, AtWak1, and LePRK1), plant hormone (AtBR1 and OsTMK), disease-resistant (OsXa21), and environmental-stress-responsive (AtRPK1) RLKs (fig. 5 ).



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Fig. 5.—Phylogenetic analysis of plant RLK multigene families. The deduced amino acid sequences of GmRLK1, GmRLK2, GmRLK3, GmCLV1A, GmCLV1B, AtRLK, and AtCLV1 (GenBank accession numbers AF244888, AF244889, AF244890, AAF59905, AAF59906, CAA16688, and AAB58929, respectively), together with other plant RLKs (Arabidopsis thaliana AtHAESA, AtER, AtBR1, AtWak1, and AtRPK1, rice OsTMK and OsXa21, Brassica BoSRK3, maize ZmRLK2, and tomato LePRK1 [GenBank accession numbers P47735, U47029, AF017056, AJ009696, U55875, Y07748, U72724, X79432, AF023165, and U58474, respectively], and rice OsLRK1 [Kim, Jeong, and An 2000]), were aligned with the Dayhoff amino acids distance matrix. A phylogenetic tree was constructed with the cluster and topology algorithms using GeneBee-Net v.2.0 (http://www.genebee.msu.ru/services/phtree_reduced.html), and the branch lengths were adjusted by the Fitch-Margoliash method with contemporary tips, version 3.572c (http://sdmc.krdl.org.sg:8080/~lxzhang/phylip/). AtHAESA, floral organ abscission; AtER, organogenesis regulation; AtBR1, brassinosteroid signal transduction; AtWak1, pathogen response; AtRPK1, environmental stress response; OsLRK1, shoot and floral development; OsTMK, gibberellin signal tansduction; OsXa21, disease resistance; BoSRK3, self-incompatibility interaction; ZmRLK2, endosperm development; LePRK1, pollen-tube elongation

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The genome of the cultivated soybean species is highly duplicated (Hadley and Hymowitz 1973Citation ; Shoemaker et al. 1996Citation ). Most of its genes are presumed to comprise multigene families (Hadley and Hymowitz 1973Citation ; Hymowitz and Newell 1980Citation ; Shoemaker et al. 1996Citation ). Soybean GmRLKs belong to a multigene family. As presented by Song et al. (1997)Citation , the evolution of the RLK family members can be envisioned by duplication and subsequent divergence of an RLK progenitor gene, and the intra- and intergenic recombination can generate new family members. We isolated and characterized three GmRLK genes: GmRLK1, GmRLK2, and GmRLK3. Sequence identities between GmRLK1, GmRLK2, and GmRLK3 range from 80% to 95%, and GmRLK2 and GmRLK3 are almost identical. Detected amino acid replacements at the conserved domains between GmRLK1 and GmRLK2/GmRLK3 suggest that GmRLK2 and GmRLK3 have undergone a paralogous evolution. Amino acid replacements in the conserved domains may contribute to functional diversity between gene family members (Ohta 1991Citation ; Schulze-Muth et al. 1996Citation ). In C. roseus RLK CrRLK1, autophosphorylation occurs predominantly on threonine residues in the kinase subdomains (Schulze-Muth et al. 1996Citation ). Mutagenesis of Thr-720 to Ala eliminates autophosphorylation and phosphorylation of ß-casein activities (Schulze-Muth et al. 1996Citation ). On the other hand, the same amino acid substitution at a different position, Thr-767 to Ala, increases ß-casein phosphorylation approximately fivefold (Schulze-Muth et al. 1996Citation ). These examples show how mutations at specific amino acid residues could alter the autophosphorylation status and/or the substrate specificity; thereby, the following signal cascades could also change, resulting in novel physiological effects. The analysis of subdomain structures may reveal the mechanisms of diversification of the catalytic functions for structurally and functionally related proteins (Lang et al. 2000)Citation .

In genomic analysis using a nine-genome-coverage BAC library, GmRLK1, GmRLK2, and GmRLK3 were localized in five regions (contigs I–V). The GmRLK regions could be easily distinguished based on DNA fingerprinting patterns. We do not know the genetic map positions of the five GmRLK regions at this time. We investigated two primitive and five advanced genotypes with 10 restriction enzymes and found no RFLPs for the GmRLK1, GmRLK2, and GmRLK3 genes. This low degree of molecular marker polymorphisms could be due to the limited genetic sources used in cultivar development in G. max species (Morgante et al. 1994Citation ; Kisha et al. 1998Citation ). The conventional RFLP method does not allow recognition of restriction fragments over 20 kb, and thus high-molecular-weight polymorphisms cannot be revealed. Our results show that genome analysis can be enhanced by BAC DNA fingerprinting, which detects segmental reorganization of restriction sites involving large fragments in the vicinities of paralogous genes.

We identified one cluster of paralogous genes, GmRLK2 and GmRLK3 in contig IV. This cluster could have evolved similarly to the RLK cluster (Lrk and Tak) in the grass family (Feuillet and Keller 1999Citation ). Lrk and Tak have arisen from a common ancestral gene by duplication followed by structural chromosomal rearrangements. While searching the sequence similarities of GmRLKs in GenBank, we identified four A. thaliana BAC clones that contain two RLK genes in each clone. The four A. thaliana BAC clones are scattered on three chromosomes: two (GenBank accession numbers AL132955 and AL133292) on chromosome III, one (GenBank accession number AL022224) on chromosome IV, and one (GenBank accession number AL021684) on chromosome V. The BAC clone containing AtRLK (GenBank accession number CAA16688), the ortholog of GmRLK1, GmRLK2, and GmRLK3, is mapped at 131.1 cM on chromosome V, close to marker mi335 (http://genome-www3.stanford.edu). Grant, Cregan, and Shoemaker (2000)Citation identified substantial synteny between A. thaliana and soybean chromosomes using conceptual translations of DNA sequences; the sequences of soybean RFLPs on linkage group soy2A had strong homologies to three A. thaliana BAC sequences from chromosomes I, IV, and V. However, GmRLK1, GmRLK2, and GmRLK3 did not localize at these syntenic regions. Previously, we mapped one of the soybean RLKs (GmCLV1); an EcoRI RFLP of GmCLV1 was assigned on linkage group H of the soybean molecular map (Yamamoto, Karakaya, and Knap 2000)Citation . In A. thaliana, AtCLV1 is mapped at 117.8 cM, close to marker m532 (1.5 kb from orf1-1) on chromosome I (Williams, Clark, and Meyerowitz 1999Citation ), suggesting that homeologous regions might also be present in linkage group H in soybean and chromosome I in A. thaliana.

Phylogenetic analysis indicates that a common ancestral GmRLK gene may have duplicated to give rise to GmRLK1, GmRLK2, and GmRLK3; GmRLK1 may have developed earlier than GmRLK2 and GmRLK3. The BAC contig analysis suggests that gene duplication was accompanied by genome rearrangement at the submegabase level. Evolutionary analysis of soybean RLK genes together with several other plant species indicates that CLV1 and RLK genes may have been generated by gene duplication from a common ancestral gene before the divergence of soybean, A. thaliana, and rice. AtCLV1- and AtRLK-related genes may have evolved later than the other plant RLKs involved in developmental, biotic, and abiotic responses. AtCLV1 is associated with shoot and floral meristem development (Clark, Williams, and Meyerowitz 1997Citation ). Thus, AtCLV1-related genes including GmRLKs and GmCLV1s might have acquired in the course of evolution more specialized functions in plant ontogeny. The presence of isotype genes in a multigene family may allow species plasticity and confer selective advantage to an organism.



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Appendix 1.—Partial sequences of soybean receptor-like protein kinase (RLK) genes. PCR primer sites are underlined and indicated with arrows. The stop codons are in boldface type. GenBank accession numbers are AF244888 (GmRLK1), AF244889 (GmRLK2), and AF244890 (GmRLK3). The EMBL alignment number is DS42558

 


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Appendix 2.—Genomic distribution of soybean receptor-like protein kinase (RLK) genes. A, Contig assembly of DNA fingerprinted BAC clones from a soybean genomic library. A soybean BAC library was screened with GmRLK1, GmRLK2, and GmRLK3 probes independently. Contigs I, II, and V contain GmRLK1; contig III contains GmRLK2; and contig IV contains GmRLK2 and GmRLK3. Five contigs do not overlap each other. The order of the contigs is arbitrarily arranged. BAC clones on the microtiter plates in the library are indicated. The sizes of BAC clones range between 56 and 300 kb. B, Identification of GmRLK1, GmRLK2, and GmRLK3 in BAC clones and genomic DNA by PCR using gene-specific primers. Lane 1, 100-bp DNA ladder; lanes 2–6, contigs I, II, and V; lanes 7–11, contig III; lanes 12–16, contig IV; lanes 17–21, genomic DNA. Lanes 2, 7, 12, and 17, GmRLK1 (IF-IR, 262 bp); lanes 3, 8, 13, and 18, GmRLK2 (IIF-IIR, 215 bp); lanes 4, 9, 14, and 19, GmRLK3 (IIIF-IIIR, 247 bp); lanes 5, 10, 15, and 20, GmCLV1A (1A3'5F-1A3'6R, 213 bp); lanes 6, 11, 16, and 21, GmCLV1B (1B3'7F-1B3'8R, 200 bp)

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Drs. J. Fletcher (Plant Gene Expression Center, Albany, Calif.), B. Krizek (University of South Carolina, Columbia, S.C.), D. Heckel (University of Melbourne, Parkville, Australia), and anonymous reviewers for critical comments. This is technical contribution number 4652 of the South Carolina Agriculture Experimental Station.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Keywords: soybean receptor-like protein kinase gene duplication paralogous CLAVATA1 Arabidopsis thaliana. Back

2 Address for correspondence and reprints: Halina T. Knap, Clemson University, 272 Poole, 50 New Cherry Road, Clemson, South Carolina 29634-0359. hskrpsk{at}clemson.edu Back


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 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication April 9, 2001.