Electron Paramagnetic Resonance (EPR) as a Tool to Study Nitric Oxide Generation in Plants

S. PUNTARULO, S. JASID, A. D. BOVERIS, M.SIMONTACCHI

Nitric Oxide in Plant Physiology

WILEY-VCH Verlag GmbH & Co. KGaA

Año: 2009; p. 17 - 30

Plants can produce and release nitric oxide (NO), especially under stress or in certain physiological processes, mainly in actively growing tissues such as embryonic axes. A distinctive EPR signal for the adduct MGD-Fe-NO (g = 2.03 and aN= 12.5 G) was detected in axes isolated from sorghum or soybean seeds. To determine whether NO is one of the possible in vivo metabolites of nitrate utilization, the EPR spectra of homogenates from both types of embryonic axes imbibed during 24 h in the presence of nitrate, were compared. On the other hand, in vivo generation of NO in plants is achieved through different enzymatic pathways employing either nitrite or arginine as substrates. Since NO was identified as a product from the activity of the enzyme nitrate reductase (NR), homogenates from axes were supplemented with MGD-Fe and the substrates for NADH-dependent NR activity and the rate of generation of NO was measured by EPR, to assess maximal on line NO generation by the enzymatic activity. The MGD-Fe-NO spectrum was recorded over a 10 min period and the increase on the peak height at 3412 G was plotted. A similar assay was developed for assessing Nitric Oxide Synthase (NOS)-like activity in embryonic axes. Thus, EPR was successfully employed to identify NO presence upon germination and to assess for NR and NOS-like activity by direct identification of the generated product, NO.