Extended Data Fig. 2: The treatment effect of Arabidopsis WT leaves with nitroxyl donors on HNO concentration.
From: Discovery of endogenous nitroxyl as a new redox player in Arabidopsis thaliana
a, The impact of a one-time treatment of Arabidopsis WT leaves with Angeli’s salt (AS) on HNO concentration. Leaves were sprayed with the following AS concentrations 0.25, 0.5, 1.0, and 10 mM, respectively, at 25 °C. HNO measurements with microsensor were carried out in the leaf extracts 10 min after spraying. HNO formation was comparable in AS concentration range of 0.5–10 mM indicating a leaf penetration limit for AS diffusion. This could be explained by the fact that plant epidermis is covered by an extracellular hydrophobic layer (cuticles), and AS is the sodium trioxodinitrate salt (Na2[N2O3]). This difference between nonpolar and polar compounds, especially during multiple treatments, was optimized using an organic surfactant (see c). The treatment with the lowest AS concentration (0.25 mM) decreased (20%) the HNO level. b, The effect of a three-time treatment of Arabidopsis WT leaves with AS on HNO concentration. Leaves were three-time sprayed in 10 min intervals with the following AS concentrations 0.25, 0.5, 1.0 mM, respectively, at 25 °C. HNO measurements with microsensor were carried out in the leaf extracts 10 min after the third spraying The maximum HNO concentration in leaf tissues three-time treated with 0.5 mM AS increased by 40% compared with one-time pretreatment (for comparison see A). HNO concentrations measured in a group of leaves no. 5–7 or only leaf no. 7 (pooled from several plants) treated with AS (0.5 mM) were comparable. c, The effect of time, temperature, and multiple treatments of Arabidopsis WT leaves with AS on HNO concentration. Leaves were sprayed once, three, or five- times in 1 or 10 min intervals, respectively, with 0.5 mM AS at 25 °C or 37 °C. HNO measurements with microsensor were carried out in the leaf extracts after 1 or 10 min after the last spraying, respectively. Angeli’s salt decomposition rate is temperature-dependent, so HNO production from 0.5 mM AS was almost 2-fold lower at 25 °C than at 37 °C, respectively. d, A comparison of various HNO donor’s efficiency. Leaves were sprayed with AS, 4-NO2-Piloty´s acid (NPA), and Cimlanod (CM) at 25 °C or 37 °C. HNO measurement with microsensor was carried out in the leaf extracts after 5, 30, 60, or 90 min, respectively. The most stable and effective HNO donating compound turned out to be CM (1.5 mM) with stabilized HNO emission in 60–90 min, after once leaf treatment at 25 °C. A similar amount of HNO released AS (1.5 mM) in a short time after five-time leaf treatment with this donor at 37 °C. Both NPA and AS at lower concentrations (0.5 mM) produced less HNO due to the rapid rate of donors decomposition. For multiple treatments, an organic surfactant (0.1% SG) to improve the surface contact between the donor solution and the leaf was applied, as presented in Extended Data Fig. 2c. The crude extracts were prepared as described in the Methods. Data are presented as mean ± s.d. of three biologically independent replicates (n = 3). (*) indicate values that differ significantly (P ≤ 0.05) from control WT. Statistical significance was assessed using two-tailed t-tests.
