In this report, we demonstrate that nitric oxide (NO) mediates the auxin response leading the adventitious root formation. A transient increase in NO concentration was shown to be required and to be part of the molecular events involved in adventitious root development induced by indole acetic acid (IAA).The discovery of signal molecules involved in the intricate network that triggers root formation remains a major goal for a large number of biotechnological procedures. Adventitious rooting involves the development of a meristematic tissue after removal of the primary root system. The plant hormone auxins promote this process through the dedifferentiation of cells to reestablish the new apical meristem. Although a variety of components of auxin transport and signal transduction were identified, the molecular mechanism underlying the initiation of new root meristems is poorly understood (Doerner, 2000; Berleth and Sachs, 2001).NO is a diffusible multifunctional second messenger first described in mammals, where it plays variable functions ranging from dilation of blood vessels to neurotransmission and defense during immune response (Gow and Ischiropoulos, 2001). Several researches have shown the presence of NO in plants and have attributed novel roles to this gas in the plant kingdom (Beligni and Lamattina, 2001a and refs. therein).Of late, and contemporary to genomics and proteomics, it is interesting to note the revival of pharmacological and surgical techniques in the field of plant developmental biology (Nemhauser et al., 2000; Reinhardt et al., 2000). In this communication, we demonstrate through pharmacological and surgical approaches that NO is required for root organogenesis.Two NO donors, sodium-nitroprussiate (SNP) and S-nitroso, N-acetyl penicillamine (SNAP), applied to hypocotyl cuttings (primary roots removed) of cucumber (Cucumis sativus) were able to mimic the effect of the auxin IAA in inducing de novo root organogenesis (Fig. 1). In addition, NO-and IAAinduced roots presented similar anatomic structure when they were analyzed by optic microscopy (not shown). This NO-mediated effect was prevented when the specific NO-scavenger carboxy-PTIO (cPTIO) was added with SNP or SNAP (Fig. 1). Result of treatments performed with different SNP concentrations confirmed that the effect was dose dependent, with a maximal biological response at 10 m SNP (Fig. 2). Within 3 d after removal of the primary root system, adventitious root development was detected in the explants treated with IAA, SNP, or SNAP. Two parameters of root growth were considered, and length and number of adventitious roots exhibit similar behavior among these treatments (Fig. 3, t test, P Ͻ 0.05). In control experiments, when hypocotyl cuttings were kept in water or in NO 2 Ϫ / NO 3 Ϫ (normal products of NO decomposition, not shown), adventitious roots emerged 4 d after primary root removal, and they reached only 22% of the length obtained from NO-or IAA-treated explants (Fig. 3). The treatment of hypocotyls with SNP or SNAP plus IAA resu...

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and L-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 6 0.2 nmol min 21 mg 21 protein in the presence of 1 mM NaNO 2 . Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 6 0.04 nmol min 21 mg 21 protein) of NO generation. Chloroplasts incubated with 1 mM Arg showed NO production of 0.76 6 0.04 nmol min 21 mg 21 protein that was not affected either by omission of Ca 21 or by supplementation with Ca 21 and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N v -nitro-L-Arg methyl ester hydrochloride and N v -nitro-L-Arg. In vitro exposure of chloroplasts to an NO donor (250 mM S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mM peroxynitrite (ONOO 2 ) led to an increase in lipid-derived radicals (34%). The effect of ONOO 2 on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO 2 treatment significantly affected both O 2 evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g-1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.

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