Plants and growth conditions
Seeds of M. truncatula cv Jemalong A17 were surface-sterilized, germinated and transformed with binary vectors via Agrobacterium rhizogenes strain Arqua1 (Quandt et al., 1993) according to Boisson-Dernier et al. (2001). Transgenic roots were selected under a Leica MZ FLIII fluorescence stereomicroscope (Leica, Wetzlar, Germany) based on the GFP signal two weeks after germination. After removing non-transgenic roots, composite plants were transferred to new plates with Fahräeus medium and 0.2 mM NH4NO3. One week after the transfer, plants were inoculated with Sinorhizobium meliloti at OD600 0.5 as described by Boisson-Dernier et al. (2001).
Hypoxia was performed by placing plants in hypoxic workstations, where the normal air was replaced with 100% N2, and closed for 24 hours, in the dark. In control conditions, plants were placed in the dark for 24 hours.
For the experiment involving A. thaliana mesophyll protoplasts, Columbia-0 (Col-0) and prt6 knockout Arabidopsis mutant seeds (Licausi et al. , 2011) were sown in a growing mixture containing soil (Spezial Substrate, Hawita Flor, Vechta, Germany) and perlite (3:1) (Agrilit, Perlite Italiana, srl) . Plants were grown at 25/23 °C day/night with a photoperiod of 12 h light (PAR 100 µmol m-2s-1) for 3 weeks.