The overexpression of MtN5, an early nodulin, in A. thaliana confirms its role in ROS detoxification

MtN5 is a non-specific Lipid Transfer Protein (nsLTP), expressed in the epidermis and in root hairs after rhizobial infection, and in nodule primordia and nodules at a later stage. MtN5 is an early nodulin required to successfully establish the symbiotic relationship between Medicago truncatula and Sinorhizobium meliloti. Thanks to its lipid binding capacity, MtN5 probably participates in the re-modelling of membranes during rhizobial infection, or in the signalling between the host cells and rhizobia. To better understand the role of MtN5 outside of symbiosis facilitation, we ectopically over expressed MtN5 in Arabidopsis thaliana and focused on its role during abiotic stress. Under normal conditions, A. thaliana MtN5 overexpressing lines (OE1 and OE2) grew just like WT plants, while differences were found when stresses were imposed: for salt stress (150 mM NaCl), OE1 and OE2 lines showed higher sensitivity compared to WT resulting in shorter roots; conversely, osmotic stress (200 mM mannitol) did not provoke a significant response in root length compared with WT root length; lastly, ABA treatment (5 µM) caused a reduction in WT root growth compared to roots grown in the untreated media, while OE lines were not impaired in their root growth by the same stress. Among the transcripts that were found to be down-regulated in M. truncatula MtN5 silenced lines, there was Medtr1g088910, coding for glutaredoxin-C1 and is involved in ROS detoxification. It was found to be more expressed in nodules than in roots, suggesting a specific involvement in the symbiosis process. We identified its orthologues in A. thaliana, namely ROXY4 and ROXY5 (AT3G62950, and AT3G62950 respectively) and will investigate their patterns of expression to better understand their response to N starvation and oxidative stress. Moreover, a nodulation assay was performed on a M. truncatula glutaredoxin-C1 knock-out line. WT and KO lines were inoculated with Sinorhizobium meliloti. The KO line showed a significantly lower number of nodules compared to WT plants. There is evidence that ROS are actively involved as signals in the nodulation process in M. truncatula and it is therefore crucial to unravel the gene network controlling it to develop new strategies to improve N fixation in plants.