We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus. Access to the Lotus TILLING facility can be obtained through

Arabidopsis thaliana has, in conjunction with A. arenosa, developed into a system for the molecular analysis of alloplolyploidy. However, there are very few Arabidopsis lines available to study autopolyploidy. In order to investigate polyploidy on a reliable basis, we have optimised conventional methodologies and developed a novel strategy for the rapid generation and identification of polyploids based on trichome branching patterns. The analysis of more than two dozen independently induced Arabidopsis lines has led to interesting observations concerning the relationship between cell size and ploidy levels and on the relative stability of tetraploidy in Arabidopsis over at least three consecutive generations. The most important finding of this work is that neo-tetraploid lines exhibit considerable stability through all the generations tested. The systematic generation of tetraploid collections through this strategy as well as the lines generated in this work will help to unravel the consequences of polyploidy, particularly tetraploidy, on the genome, on gene expression and on natural diversity in Arabidopsis.

SUMMARYArbuscular mycorrhiza (AM) fungi form nutrient-acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM-defective mutants via a microscopic screen of an ethyl methanesulfonate-mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild-type-like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.

The subtilisin-like serine protease SbtM1 is strongly and specifically induced during arbuscular mycorrhiza (AM) symbiosis in Lotus japonicus. Another subtilase gene, SbtS, is induced during early stages of nodulation and AM. Transcript profiling in plant symbiosis mutants revealed that the AM-induced expression of SbtM1 and the gene family members SbtM3 and SbtM4 is dependent on the common symbiosis pathway, whereas an independent pathway contributes to the activation of SbtS. We used the specific spatial expression patterns of SbtM1 promoter β-d-glucuronidase (GUS) fusions to isolate cis elements that confer AM responsiveness. A promoter deletion and substitution analysis defined two cis regions (region I and II) in the SbtM1 promoter necessary for AM-induced GUS activity. 35S minimal promoter fusions revealed that either of the two regions is sufficient for AM responsiveness when tested in tandem repeat arrangement. Sequence-related regions were found in the promoters of AM-induced subtilase genes in Medicago truncatula and rice, consistent with an ancient origin of these elements predating the divergence of the angiosperms.

The coordinated control of Ca2+ signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca2+ influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function CNGC mutation (brush) in Lotus japonicus resulting in a leaky tetrameric channel. brush resides in a cluster of redundant CNGCs encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic brush mutation impaired root development and infection by nitrogen-fixing rhizobia. The brush allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the brush phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.

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