The dynamic distribution of NO and NADPH-diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO-generating system in auxin-induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole-3-butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N-G-nitro-L-arg-methyl ester (L-NAME). Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate and survey of NADPH-diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH-diaphorase signals were specifically located in the adventitious root primordia in the basal 2-mm region (as zone I) of both control and IBA-treated explants. With the development of root primordia, NO and NADPH-diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH-diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH-diaphorase activity similar to that in zone I were also observed in the basal 2-4-mm region (zone II) of IBA-treated explants, but neither NO nor NADPH-diaphorase signals were detected in this region of the control explants. L-NAME and c-PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH-diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA-induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS-like enzyme.