Plants of the legume family have the unique ability of being able to form nitrogen fixing root nodules in symbiotic association with soil bacteria generally known as rhizobia. Success of the legume-Rhizobium symbiosis requires the coordinated action of certain bacterial as well as plant genes. While all bacterial genes involved in symbiotic nitrogen fixation have been identified so far, plant genes required for the establishment and control of nodulation are only just being characterised. The dissection of nodule organogenesis using forward genetics resulted in the identification of several plant loci involved in nodule development and maintenance. The nitrate tolerant symbiosis (NTS- 1) locus of soybean (Glycine max (L.) Merr.) is one such locus, which was identified following ethyl methyl sulphonate (EMS) mutagenesis. Plants carrying the nts-1 mutation are defective in the systemic autoregulatory mechanism, which governs nodule development. Such mutants nodulate profusely when inoculated with Bradyrhizobium even in the presence of high nitrate. In the effort to understand the molecular mechanisms that underlie systemic autoregulation of nodulation (AON), this research was carried out with the aim of map-based cloning and characterisation of the NTS-1 locus for which there was no known biochemical product.
Map-based cloning of the NTS-1 locus was aided by the use of FN37, a Glycine soja supemodulation mutant isolated by fast neutron mutagenesis. Reciprocal grafts of FN37 revealed systemic shoot control of autoregulation similar to that of EMS mutants nts382 and ntsl007. BAC end and marker analysis of the southern and northern genomic regions flanking NTS-1 confirmed a large deletion in FN37 within the same region of soybean linkage group H as the supemodulation(nts-1) locus. Although the exact size of the FN37 deletion is unknown, it was estimated to be approximately 460 kb based on mapping and sequencing data FN37 is the first nodulation mutant isolated by fast neutron mutagenesis. Essentially it proved useful in the identification and subsequent isolation of NTS-1.
NTS-1 candidate gene GmCLV1B was isolated following a series of chromosome walks. Codon-altering mutations identified within the coding sequence of GmCLV1B in several mutant (nts) alleles confirmed isolation of the NTS-1 locus, which was renamed "Glycine max nodule autoregulation receptor kinase" (GmNARK) in accordance with its biochemical and physiological functions. GmNARK is strongly homologous to another soybean gene GmCLV1A with an unknown function. The GmNARK gene structure consists of two exons and a single intron. The deduced GmNARK sequence encodes a putative receptor-like protein kinase similar to Arabidopsis CLAVATAl (AtCLVl) that functions in regulating meristem development in shoot apices. Eleven putative serine/threonine phosphorylation sites were predicted in the GmNARK kinase domain.
Transcript expression profiling showed that GmNARK is expressed in the stem, leaves, root tips, nodules and shoot apical meristem (SAM). GmNARK was most highly expressed in stem and leaf tissue in contrast to very low expression in the SAM. Overexpression of GmNARK and GmCLV1A in the Arabidopsis calve 1-1 mutant did not complement the clv1 phenotype. GmNARK is orthologous to other NARK genes HAR1and SYM29 from Lotus japonicus and pea. The NARKs were grouped in the same phylogenetic clade as AtCLVl indicating evolutionary and possible functional similarities with AtCLVl. An 8 bp motif (TAGTGGAT) involved in negative regulation of the Brassica napus extA extensin gene was identified in the GmNARK promoter region.
The isolation of GmNARK has provided crucial information regarding the biochemical nature of the NTS-1 locus governing systemic autoregulation and has opened the way to further characterise signal transduction cascades both upstream and downstream of GmNARK. Complete elucidation of the GmNARK signalling pathway will provide better understanding regarding systemic control of nodule development, a key process that is absent in the model plant Arabidopsis. Published data that AtCLVl associates with other proteins forming a receptor complex currently serves as a model for understanding GmNARK signalling.