Unravelling the role of ASN1 gene in the model legume Lotus japonicus
Asparagine constitutes the major form of nitrogen translocated within Lotus japonicus plants. Previous work had indicated that the different genes encoding for asparagine synthetase (ASN genes) show differential patterns of expression in different organs from plants grown under either symbiotic (S) or non-symbiotic (NS) conditions.  In this work we use knock out mutants deficient in LjASN1 gene, which is the most highly expressed ASN gene in NS conditions, but much less expressed under S conditions. The analysis of two different lines of LORE 1 Ljasn1 homozygous mutant plants grown in the presence complete nutrient medium (NS) or with Mesorhizobium loti (S) was comparatively examined with respect to the WT. The results obtained indicate that a much higher biomass was produced in Ljasn1 mutants grown under NS conditions compared to the WT, while little difference was observed in the mutant plants compared to the WT under S conditions. The results point out a relationship between the deficiency of the ASN1 gene and growth of L. japonicus plants mainly under NS conditions. Further metabolomic analysis revealed that Ljasn1 mutant plants are quite distinct to WT plants when grown under NS conditions, but not under S conditions, in accordance with previous results. Interestingly, asparagine level was considerably reduced in Ljasn1 mutant plants compared to the WT when plants were grown under NS but not under S conditions, thus confirming the crucial relevance of ASN1 in the asparagine levels found in the plants under NS conditions. The results from whole metabolomics analysis, together with other obtained by RNA seq analysis, indicate that ASN1 deficiency produce important changes in the C/N balance and metabolite allocation of  L. japonicus plants resulting in a higher biomass content. Overall design: A comparative RNAseq analysis of WT and of Ljasn1_2 mutant, KO for the LjAsn1 gene encoding for one isoform of asparagine synthetase was carried out.Shoots, roots or nodulated roots were harvested from plants grown either with ammonium nitrate or under symbiotic conditions. WT and mutant tissues were harvested after 4 hours of the beginning of the light period after 40 days of growth under symbiotic or non-symbiotic (provided with ammonium nitrate) conditions.