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Long-Distance Signaling in Nodulation Directed by a CLAVATA1-Like Receptor Kinase

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Science  03 Jan 2003:
Vol. 299, Issue 5603, pp. 109-112
DOI: 10.1126/science.1077937

Abstract

Proliferation of legume nodule primordia is controlled by shoot-root signaling known as autoregulation of nodulation (AON). Mutants defective in AON show supernodulation and increased numbers of lateral roots. Here, we demonstrate that AON in soybean is controlled by the receptor-like protein kinase GmNARK (Glycine maxnodule autoregulation receptor kinase), similar toArabidopsis CLAVATA1 (CLV1). Whereas CLV1 functions in a protein complex controlling stem cell proliferation by short-distance signaling in shoot apices, GmNARK expression in the leaf has a major role in long-distance communication with nodule and lateral root primordia.

Multicellular organisms need to control the proliferation of pluripotent stem cells, also referred to as meristematic cells in the apices, cambium, and pericycle of flowering plants. Because organ differentiation of plants is predominantly postembryonic and does not involve cell migration, plant stem cells need to be controlled by short- and long-distance signals to achieve equilibrium between cell proliferation and differentiation. The role of short-distance signaling in plant development has been more extensively researched, and some of the key genes involved have been identified (1–8).

Legume nodulation is important in supplying nitrogen to ecological and agricultural systems. Nodule meristems form in response to mitogenic signals from symbiotic bacteria called rhizobia (6), but nodule proliferation is restricted by autoregulation of nodulation (AON) (9–12). Mutations affecting nodule meristems have been readily identified, including ones that confer supernodulation as a result of a defect in AON (Fig. 1A).

Figure 1

Cloning of GmNARK. (A) Wild-type and extreme supernodulation (AON-defective) phenotypes. (B) Genetic (G. max nts246 ×G. soja CPI 100070 mapping population) and physical maps of soybean NTS-1 region. Deletion in FN37 mutant is shown in light blue. Flanking markers pUTG132a and UQC-IS1 were used as anchor points to isolate BAC clones and build contigs. Dashed lines show microsynteny to Arabidopsis chromosomes 2 and 4. (C) Putative protein structure of GmNARK: SP, signal peptide; LRR, leucine-rich repeats (violet circles); TM, transmembrane domain. Mutations resulting in either nonsense or amino acid changes, the location of the original AFLP product (UQC-IS5) derived from BAC75M10, and the intron (arrow) are shown. (D) GmNARK (GmClv1B) and GmClv1Aare orthologs of CLAVATA1. The phylogenetic tree was constructed for a selection of related proteins with ClustalX version 1.81 and Bootstrap analysis. All of these predicted proteins are fromArabidopsis, except where otherwise noted. The three most closely related receptor-like kinases to CLAVATA1 inArabidopsis, designated RLK-A [GenBank identification number (GI): 15239123], RLK-B (GI: 15229189), and RLK-C (GI: 15235366), fall into a phylogenetic clade separate from that containing CLAVATA1, GmNARK, and GmCLV1A. Two receptor-like kinases designated RLK-D [At2g21480 in (B); GenBank accession number NP_179743] and RLK-E [At4g39110 in (B); GenBank accession number NP_195622], corresponding to the Arabidopsisregions syntenic with the soybean GmNARK region (B), were unexpectedly more distantly related to GmNARK. Other distantly related proteins included in the phylogenetic analysis were CLAVATA2 (GI: 6049563), the brassinosteroid receptor BRI1 (GI: 2392895), the auxin response protein PINOID (GI: 7208442), the tomato disease resistance proteins CF-2 (GI: 1184075) and HCR2-5D (GI: 3894393), and the Medicago sativa NORK (GI: 21698781), the M. truncatula NORK (GI:21698783), and the Lotus japonicus SYMRK (GI: 21622628) proteins required for root nodule formation. The scale bar is an indicator of genetic distance based on branch length.

Allelic supernodulating (nts) mutants of soybean were first isolated by EMS (ethylmethane sulfonate) mutagenesis (11–13). These mutants altered at theNTS-1 locus also develop more lateral roots, leading to a bushy root system in the absence of nodulation, and reduced root growth in the presence of prolific nodulation. Subsequently, additional mutations in the NTS-1 locus were induced chemically or by fast neutrons (14–16). Isolation of mutants in other legumes confirmed the generality of AON (17–19), and reciprocal grafting of supernodulating and wild-type genotypes showed that long-distance signaling was involved and that the leaf genotype controlled proliferation of nodule primordia (20–22).

To elucidate the mechanisms of this long-distance signal exchange, we used map-based cloning to isolate the NTS-1locus. Mutant alleles were mapped to soybean linkage group H close to restriction fragment length polymorphism (RFLP) marker pA132. A subclone of pA132, pUTG132a, was placed 0.7 cM from NTS-1 in a F2 population of nts382 (G. maxBragg) × G. soja (PI468.397) (23,24) and 1.3 cM from NTS-1 in a G. max nts246 × G. soja CPI 100070 population (25). nts382 and nts246 were identified in our original mutant screen (11). Amplified fragment length polymorphism (AFLP) marker UQC-IS1 also flankedNTS-1 1.9 cM away (Fig. 1B) (25). UQC-IS1 was the closest of 11 AFLP markers shown by bulk segregant analysis and genetic mapping to be linked to NTS-1 (25).

Bacterial artificial chromosome (BAC) clones derived from a soybean PI437.654 library (26) were isolated by filter-hybridization to pUTG132a and UQC-IS1, and were verified to contain either pUTG132a or UQC-IS1 by sequencing each marker from the respective BAC clone. Both the pUTG132a and UQC-IS1 BAC contigs were oriented relative to NTS-1 by mapping polymorphic BAC ends on F2 recombinants (Fig. 1B).

Confirmation of mapping was aided by the fast neutron mutantFN37 (16). Physical mapping of markers and complete BAC sequencing of BAC171O7 (135 kb) (Fig. 1B) showed that this mutant contains a chromosomal deletion in the NTS-1region. The southern and northern deletion breakpoints were localized within the BAC171O7 sequence and close to marker UQC-IS4, respectively (Fig. 1B). Arrangement of putative open reading frames from BAC171O7, sequenced BAC ends, and markers in the NTS-1 region showed contiguous microsynteny to Arabidopsis chromosomes 2 and 4 (Fig. 1B). Seven genes (three from the northern contig and four from the southern contig) were syntenic between the NTS-1 region and Arabidopsis. BAC92D22 contained three expressed sequence tags, highly syntenic with Arabidopsis chromosome 2, but was not demonstrated to overlap with either the northern or southern contig.

For the UQC-IS1 contig, UQC-IS2 (southern end of BAC 95P14) mapped 1.2 cM away from the locus (Fig. 1B). UQC-IS2 was used to identify additional BAC clones, of which BAC3K21 was demonstrated to extend toward NTS-1 by AFLP fingerprinting. BAC-end UQC-IS3 was cloned and used to identify additional BAC clones, of which BAC129E8 was shown by AFLP fingerprinting to extend further towardNTS-1. BAC-end UQC-IS4 from BAC129E8 was cloned and used to select BAC75M10, which was AFLP fingerprinted in comparison to BAC129E8. One AFLP marker called UQC-IS5, 650 base pairs (bp) in length and located within BAC 75M10, was cloned and sequenced, revealing complete identity to a known gene called GmCLV1B(27). This gene encodes a predicted protein showing 75% amino acid similarity to the shoot meristem– expressed CLAVATA1 of Arabidopsis(4). clv1 mutants ofArabidopsis have increased numbers of undifferentiated stem cells in shoot and floral meristems, and extra organs within flowers (4).

GmCLV1B was a good candidate for NTS-1because it mapped to the correct region, was absent in deletion mutant FN37 (Fig. 2), and the homologous CLV1 is involved in control of cell proliferation in shoot meristems. The full-length genomic sequence of the candidate NTS-1 gene, including about 600 bp upstream of the translation start, was obtained (GenBank accession number AY166655). Given the difficulty of transforming soybean, confirmation of the gene was obtained by sequencing an allelic series rather than by complementing the mutant phenotype. Sequencing of GmClv1B from several wild-type and mutant lines (Table 1) identified changes in the coding sequence that strongly indicated it was the gene responsible for control of nodule meristem proliferation (Fig. 1). This gene was renamed GmNARK (Glycine max nodule autoregulation receptor kinase) to reflect its putative biochemical and developmental function in root nodulation.

Figure 2

Characterization of GmNARKmutations. (A) Genomic Southern analysis withGmNARK probe on G. soja CPI 100070 (CPI),G. max PS55, G. soja PI468.397 (PI468), and mutant FN37. Arrows indicate missing fragments in the deletion mutant. The arrowhead indicates duplicated fragment in CPI, PS55, and PI468, but only a single fragment inFN37. (B) Genomic PCR of several soybean genotypes, BAC75M10, and BAC112J23 withGmNARK- and GmCLV1A- specific oligonucleotide primers. (C) Nonquantitative RT-PCR analysis of GmNARK expression in G. soja [wild type (WT)] and FN37 with GmNARK-specific 3′-UTR and actin primers. PCR template was prepared with (+) and without (−) reverse transcriptase. (D) Quantitative RT-PCR analysis of expression with GmNARK-specific 3′-UTR primers in Bragg (wild type), nts1007, and nts382 leaves and shoot apical meristem (SAM) regions from nodulated plants. Values are fold increase ratios from two PCR replicates, normalization against actin, relative to wild-type meristems. Average SE was <1%. (E) SAM regions of Bragg and nts1007 plants showing similar morphology of the tunica and central zone as well as alternating leaf primordia. Bar, 65 μm.

Table 1

Mutant alleles of GmNARK. The asterisk denotes nonsense termination mutation.

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GmNARK encodes a predicted receptor-like protein kinase (RLK) composed of a 24–amino acid NH2-terminal signal peptide (MRSCVCYTLLLFIFFIWLRVATCS), an extracellular domain composed of 19 tandem copies of a 24–amino acid leucine-rich repeat (LRR), a transmembrane domain (TRVIVIVIALGTAALLVAVTVYM), and a COOH-terminal cytoplasmic kinase domain (28). A 49–amino acid island interrupts the LRR domain between repeats 11 and 12. Overall, the protein contains 15 potential N-glycosylation sites. We were able to correlate the type of mutational change with the severity of the nodulation phenotype (Table 1 and Fig. 1C). Allele nts1007 (causing a greater than 10-fold increase in nodule number) is a nonsense mutation that truncates the protein at glutamine residue 106 (Q106*), eliminating most of the LRRs and the entire protein kinase domain. The Q106* mutation was confirmed in the Australian variety PS55, carrying the nts1007 allele. Alleles nts246,en6500, and nts382, conferring extreme phenotypes almost identical to nts1007, also truncate the protein by nonsense mutations; nts246 (K115*) is located immediately downstream of nts1007, also in the LLR domain;en6500 (K606*) is immediately upstream of the transmembrane domain; and nts382 is in the kinase domain (Q920*). The deletion mutant FN37 also has an extreme supernodulation phenotype. Because all five mutations (FN37 deletion, three receptor nonsense mutations, and a kinase nonsense mutation) showed indistinguishable extreme supernodulation, the loss of the kinase activity is sufficient to confer the extreme phenotype. In contrast, the weak allele nts1116 conferred two- to threefold increased nodulation. It is caused by a transition of valine to alanine at position 837, also in the kinase domain (Fig. 1C). Thus, gene identification was confirmed by the characterization of six independent mutant alleles, in which the predicted impact of the molecular alteration of the protein is precisely reflected in the severity of the phenotype. We confirmed that gene structure and amino acid sequence are conserved in wild-type soybean cultivars Clark, Williams, Bragg,G. soja PI468.397, and G. soja CPI 100070. Some silent mutations exist in the wild-type G. soja lines.

In contrast to CLV1, which is present as a single copy in Arabidopsis, in soybean GmNARK(GmCLV1B) is strongly homologous to a duplicated gene called GmCLV1A (27). GmNARKand GmCLV1A were previously investigated in a study of stem fasciation (27). Both genes were reported to be wild type in fasciated soybean mutants but linked to pA381-1 (27), a RFLP marker in the vicinity of NTS-1(24). The GmCLV1A gene differs fromGmNARK by only ∼10% at the nucleotide level, but a greater sequence divergence in the 3′-untranslated regions (UTRs) allowed gene-specific amplification (27). Interpretation of polymerase chain reaction (PCR) and Southern analyses was aided by the availability of deletion mutantFN37, which lacks GmNARK and severalNTS-1-linked markers (Figs. 1B and 2, A and B).GmNARK, but not GmCLV1A, is located on overlapping BAC clones BAC75M10 and BAC112J23 (Fig. 2B).GmCLV1A is only weakly detected in Southern analysis ofFN37, presumably through the presence of a duplicated copy of GmCLV1A elsewhere in the genome.

Reverse transcription (RT)–PCR analysis with GmNARK3′-UTR–specific oligonucleotide primers was also used to determine tissue-specific transcript levels. Whereas Arabidopsis CLV1 expression is restricted to the shoot apical meristem (SAM) (4), GmNARK is expressed in nodulated roots and shoots of the wild type (Fig. 2, C and D). NoGmNARK transcript was detected in FN37 (Fig. 2C). Quantitative RT-PCR also revealed that leaf GmNARKtranscript levels are substantially higher than those in the SAM tissue (Fig. 2D) for wild type, nts1007, and nts382. Such expression of GmNARK is consistent with a primary role of the leaf in AON (20–22).

GmNARK contains a single intron (465 bp) in the kinase domain, in precisely the same position as the intron (79 bp) inCLV1 (4). The similarity of gene structure and protein sequence suggests that GmNARK shares functional and evolutionary similarities with CLV1 (Fig. 1D). CLV1 controls stem cell proliferation in shoot meristems, and mutations of CLV1 lead to apical and floral meristem changes, whereas GmNARK functions in the leaf and exerts long-distance control of nodulation with no detectable SAM (Fig. 2E), floral, or leaf phenotypes. Also, GmNARK displays differential tissue-specific expression to CLV1. It is therefore likely that GmCLV1A is the immediate functionalCLV1 ortholog and that GmNARK is a duplicated version of CLV1, but with a different expression pattern and divergent function involved in long-distance control of nodulation.

The discovery of a receptor-like protein kinase as part of the signaling circuit for AON opens the possibility of characterizing associated long-distance signals in plant development. BecauseGmNARK is closely related to CLV1, one can presume that both upstream and downstream signal transduction are broadly similar in their mode of action. It is therefore likely that the predicted extracellular LRR domain of GmNARK interacts with another protein similar to Arabidopsis CLV2 and an extracellular peptide similar to Arabidopsis CLV3 (1,5). Other proteins involved in signal transduction have also been shown to interact with CLV1 in Arabidopsis (5).

Intriguingly, GmNARK is most similar to CLV1, whereas two receptor-like kinase genes in the regions on chromosomes 2 and 4 of Arabidopsis syntenic with the soybeanNTS-1 region are much more distantly related (Fig. 1D). There is no synteny between the NTS-1 region of soybean and the vicinity of CLV1 (29). One possible explanation for this finding is that a localized gene recombination or conversion-like event may have occurred in evolution involving theCLV1 ortholog and another receptor-like kinase gene, such as to change the chromosomal location of CLV1 in eitherArabidopsis or soybean.

Other receptor-like kinases have been shown to participate in environmental sensing—for example, in the perception of hormones, pathogens, symbionts, or cellular interactions (1–3, 7–8). The discovery of a divergent Arabidopsis CLV1 ortholog in soybean effecting long-distance nodulation control extends this spectrum of activities to cell division events in a distal organ that are first sensed, then homeostatically controlled.

Our findings suggest evolutionary mechanisms for the development of the root nodule symbiosis. Duplication of genes followed by divergence in function is a common theme in evolution (30). Ancestral duplication of a gene controlling stem cell proliferation in the SAM may have led to a variant mechanism in which shoot control of cell proliferation is extended to root tissue. Research in legumes into CLAVATA-related signaling will undoubtedly facilitate the understanding of key developmental processes such as nodulation that are absent in the model plant Arabidopsis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1077937/DC1

Materials and Methods

Tables S1 and S2

References

  • * Present address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829, Köln, Germany.

  • These authors contributed equally to this work.

  • Present address: Zoology and Entomology, School of Life Sciences, The University of Queensland, Brisbane, Australia.

  • § Conjoint member of Institute of Molecular Bioscience, The University of Queensland.

  • || To whom correspondence should be addressed. E-mail: b.carroll{at}mailbox.uq.edu.au

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