Molecular analysis of the dihydrodipicolinate synthase gene family of leguminous plants

DHDPS is a key enzyme in the aspartate-derived lysine biosynthesis pathway in plants and negatively feedback-regulated by lysine itself. Recently, DHDPS isoforms with novel regulatory properties were demonstrated in the model legume Medicago truncatula and hypothesized to be involved in abiotic and biotic stress responses. Based on the findings in M. truncatula, the main objective was to investigate further to what extent DHDPS isozymes with potentially novel molecular properties are present in legumes and how we can exploit this knowledge, including novel biofortification strategies.

To achieve this, the research was layered in three-fold. One, was an in silico analysis including a phylogenetic - and promoter analysis in land plants and legumes respectively, preceding a large scale RNA-seq re-analysis in Lotus japonicus, M. truncatula and G. max. Two, we explored the naturally occurring genetic variation of the DHDPS genes in the model legume M. truncatula, in addition to a DHDPS candidate gene-association analysis with root growth under normal or salt stress conditions and DHDPS activity or sensitivity to Lys feedback inhibition as our phenotypes of interest. Three, a mutation analysis of various plant DHDPS isozymes, measuring the effect of specific single amino acid changes on lysine feedback inhibition-sensitivity of the purified proteins in vitro. The in silico analysis established the existence of a legume-specific class of DHDPS, hereby termed as the DHDPS B-type, distinguishable from the DHDPS A-type commonly present in all land plants. In addition, we provide strong evidence that the DHDPS gene family is linked to specific abiotic and biotic stress responses in the plant. An interesting finding is the upregulation of a subgroup of DHDPS B-type genes specific upon root interaction with Glomus intraradices, a symbiotic arbuscular mycorrhizal fungus. Mutation of the highly conserved histidine to a lysine at position 80 of the protein (Nicotiana sylvestris numbering) renders the sensitive A-type DHPDS, insensitive to lysine feedback inhibition, however reducing hereby the total activity of the protein. This mutation has high potential of use in creating crops with a higher free lysine content.

Together these results contribute significantly to the knowledge of the genetics and molecular properties of the DHDPS gene family in leguminous plants.