• hypoxia
• flooding
• legumes
• Medicago
• rhizobia
• symbiosis
• nodulation
• nitrogen fixation

Most legumes are able to evolve a symbiotic relationship with nitrogen-fixing bacteria. In the symbiosis, bacteria are localized within nodules, root organs characterized by a microoxic environment.
Over the past six decades, floods have increased in many ecosystems. Flooding is a condition which limits oxygen availability.
Little information is available about how the processes of nodulation and nitrogen fixation are affected in a condition of low environmental oxygen.
First, we studied the effect of hypoxia on nodulation in Medicago sativa. Since hypoxia has been shown to arrest nodule formation, we further investigated this process using a M. truncatula pMtENOD11–gusA transgenic line and we observed that hypoxia induces an arrest in the developmental program of the nodule, which is normally resumed after reoxygenation. qPCR and RNA-seq analyses revealed that hypoxia could be altering the expression of nodulation genes.
To mimic what happens in fields we tested the effect of soil waterlogging on nodule development and bacterial infection efficiency, both of which resulted to be negatively affected by this condition. In addition, we observed that the nitrogenase activity is significantly inhibited by waterlogging.
Furthermore, we designed a low oxygen reporter based on the HRPE (hypoxia responsive promoter element) promoter and we used it to perform a transient transformation. Sectioned transgenic nodules revealed the activation of this promoter in the hypoxic core.
Finally, we designed the setting to obtain oxygen signalling mutants in Medicago through CRISPR-Cas9.
This study allowed us to find out that external hypoxia is able to arrest nodule organogenesis, as well as having an effect on nitrogen fixation.