The major feature of the plant-growth-promoting bacteria Azospirillum brasilense is its ability to modify plant root architecture. In plants, nitric oxide (NO) mediates indole-3-acetic acid (IAA)-signaling pathways leading to both lateral (LR) and adventitious (AR) root formation. Here, we analyzed aerobic NO production by A. brasilense Sp245 wild type (wt) and its mutants Faj009 (IAA-attenuated) and Faj164 (periplasmic nitrate reductase negative), and its correlation with tomato root-growth-promoting effects. The wt and Faj009 strains produced 120 nmol NO per gram of bacteria in aerated nitrate-containing medium. In contrast, Faj164 produced 5.6 nmol NO per gram of bacteria, indicating that aerobic denitrification could be considered an important source of NO. Inoculation of tomato (Solanum lycopersicum Mill.) seedlings with both wt and Faj009 induced LR and AR development. In contrast, Faj164 mutant was not able to promote LR or AR when seedlings grew in nitrate. When NO was removed with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), both LR and AR formation were inhibited, providing evidence that NO mediated Azospirillum-induced root branching. These results show that aerobic NO synthesis in A. brasilense could be achieved by different pathways and give evidence for an NO-dependent promoting activity on tomato root branching regardless of bacterial capacity for IAA synthesis.

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Azospirillum and other related plant growth-promoting rhizobacteria produce several phytohormones and signaling molecules. Among them, nitric oxide is now recognized as a key second messenger in plants and in benefi cial and pathological plant-microorganism interactions switching on and off different processes. Nitric oxide has been associated to the signaling cascades leading to lateral and adventitious root development induced by Azospirillum in tomato, in the Azospirillum biofi lm formation, and in the nodule development in rhizobia-legume symbiosis. As a central component of N cycle, nitric oxide is produced and consumed in different metabolic pathways such as denitrifi cation and nitrifi cation, and it is closely related to other N compound like nitrate and nitrite. The emerging importance of nitric oxide in the biology of bacteria-plant relation is a challenge for understanding the molecular and chemical basis underpinning the nitric oxide actions in the association of plant growth-promoting bacteria with roots. In this chapter we describe several techniques that allow detecting and quantifying endogenously produced and exogenously applied nitric oxide in bacteria cultures and inoculated plants, including real-time and/or in situ nitric oxide production. The most used methods-Griess assay, electron paramagnetic resonance, fl uorescent probes, and electrochemical sensors-are described with detailed protocols, discussing their advantages and drawbacks. In addition, we remark factors affecting nitric oxide production like growth conditions, metabolic inhibitors, and others. Finally, pharmacological, genetic, and histochemical strategies to study the role of nitric oxide in the association of Azospirillum with plant roots are presented with examples and methodological procedures.