Extended Data Fig. 7: Kinetics of endogenous HNO and •NO formation in WT Arabidopsis leaves pretreated with cellular reductants.
From: Discovery of endogenous nitroxyl as a new redox player in Arabidopsis thaliana
a, The effect of reducing conditions induced in leaves by ascorbic acid (1 mM AA), b, salicylic acid (1 mM SA) and c, hydrogen sulphide (1 mM H2S) on HNO/•NO generation. Enrichment of leaves with pseudo-aromatic alcohols such as AA and SA (a, b) resulted in a time-dependent rise in the HNO level measured in vivo by electrodetection in leaf extracts. However, ascorbate as a diol may react in vitro approximately twenty times faster than SA being a phenol63,64. The proposed mechanism involves a nucleophilic attack to •NO by the alcohols, coupled with proton transfer and subsequent decomposition of the thus-produced radical to yield HNO (Supplementary Table 3). Leaf pretreatment with H2S (c) also resulted in a significant increase in HNO production. The signal accelerated during the first minutes reflecting the fast nature of the reaction between H2S and •NO to produce HNO (k = 104 M−1s−1)65. However, there is a more complicated mechanism than a simple bimolecular reaction between •NO and H2S/HS− (Supplementary Table 3), but the response of the successful attack by two NO molecules to this gasotransmitter (H2S) produces HNO4,65. In this context the reported redox potential, E´(NO, H+/ HNO), at physiological •NO and HNO concentration is between −0.3 and 0 V at pH = 7.4. Thus, the one-electron reduction process for NO is feasible under biological conditions and could be promoted by the analyzed reductants with reduction potentials around −0.3 to −0.5V4,65. Data are presented as mean ± s.d. of three biologically independent replicates (n = 3). (*) indicate values that differ significantly (P ≤ 0.05) from control (untreated) WT leaves. Statistical significance was assessed using two-tailed t-tests.
