A detailed microscopical analysis of the morphological features that distinguish different developmental stages of nodule organogenesis in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants was performed, to provide the necessary framework for the evaluation of altered phenotypes of L. japonicus symbiotic mutants. Subsequently, chemical ethyl methanesulfonate (EMS) mutagenesis of L. japonicus was carried out. The analysis of approximately 3,000 M1 plants and their progeny yielded 20 stable L. japonicus symbiotic variants, consisting of at least 14 different symbiosis-associated loci or complementation groups. Moreover, a mutation affecting L. japonicus root development was identified that also conferred a hypernodulation response when a line carrying the corresponding allele (LjEMS102) was inoculated with rhizobia. The phenotype of the LjEMS102 line was characterized by the presence of nodule structures covering almost the entire root length (Nod++), and by a concomitant inhibition of both root and stem growth. A mutation in a single nuclear gene was shown to be responsible for both root and symbiotic phenotypes observed in the L. japonicus LjEMS102 line, suggesting that (a) common mechanism(s) regulating root development and nodule formation exists in legumes.

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with 15 N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.

The Euglenophyceae chloroplast was acquired when a heterotrophic euglenoid engulfed a green alga and subsequently retained the algal chloroplast, in a process known as secondary endosymbiosis. Since this event, Euglenophyceae have diverged widely and their chloroplast genomes (cpGenomes) have as well. Changes to the cpGenome include extensive gene rearrangement and the proliferation of introns, the analyses of which have proven to be useful in examining cpGenome changes throughout the Euglenophyceae. The Euglenales fall into two families, Euglenaceae and Phacaceae. Euglenaceae contains eight genera and at least one cpGenome has been published for each genus. Phacaceae, on the other hand, contains three genera, none of which have had a representative chloroplast genome sequenced. Members of this family have many small disk-shaped chloroplasts that lack pyrenoids. We sequenced and annotated the cpGenome of Phacus orbicularis in order to fill in the large gap in our understanding of Euglenophyceae cpGenome evolution, especially in regard to intron number and gene order. We compared this cpGenome to those of species from both the Euglenaceae and Eutreptiales of the Euglenophyceae phylogenetic tree. The cpGenome showed characteristics that were more derived than that of the basal species Eutreptia viridis, with extensive gene rearrangements and nearly three times as many introns. In contrast, it contained fewer introns than all but one of the previously reported Euglenaceae cpGenomes, had a smaller estimated genome size, and shared greater synteny with two main branches of that family.