During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

Temperature-dependent magnetic susceptibility and multi-frequency EPR (9.4, 34.5, 94 and 188 GHz) spectroscopic measurements have been carried out together with an X-ray study at 100 K to study [Fe(DMSO)₆](NO₃)₃. The iron(III) ion remains high-spin (S = 5/2) in the temperature range studied, therefore, the EPR data were interpreted using the conventional S = 5/2 spin Hamiltonian. A full analysis of EPR spectra at 95 GHz of a powdered sample at 290 K revealed that they are extremely sensitive to D and E values. The zfs parameters were precisely determined: D = + 0.1730 cm⁻¹, E = 0.00 cm⁻¹ and λ = |E/D| = 0.00. A sequence of the spectra neatly shows that the compound has a clear magnetic dependence on temperature. The study at 5 K, showed that the zfs parameters increase: D = + 0.1970 cm⁻¹, E = 0.017 cm⁻¹ and λ = |E/D| = 0.086. These data indicate that as the temperature decreases the D tensor increases slightly showing an increase in the rhombicity. These results confirm that |2D| ≅hν at X-band in this case. Additionally, it has been shown by X-ray crystal analysis of [Fe(DMSO)₆](NO₃)₃ at 100 K that this is involved in a hydrogen bonding network, consisting of C–H⋯O interactions between the nitrate anions and the methyl groups of the coordinated DMSO molecules, thus suggesting that the differences found in the spectroscopic parameters D and E at different temperatures must be due to these supramolecular interactions.