Nitric oxide oxidative production from tryptophan derivatives of the indole-3-acetic acid biosynthesis pathway in plants

Abstract

Nitric oxide (NO) is a small molecule that possesses a wide range of physiological functions in living organisms. In plants, NO has been revealed to be involved in many physiological functions, such as germination, flowering, leaf senescence, and in the response to environmental stresses. In plants, NO production has been well characterised in reductive routes, as the nitrate reductase pathway. Since plants grown axenically with NH4+ as the sole source of N have exhibited contents of NO2− and NO3−, this evidences the existence of a metabolic pathway for the oxidative production of NO. The presence of nitric oxide synthases (NOS) in bacteria, fungi, and animals has given rise for an intense search for a NOS-like enzyme in plants. Oximes (R1R2C=NOH) are important compounds connecting the general and specialised metabolisms that have been reported to release NO in basic environments. In particular, the plant oxime indole-3-acetaldoxime (IAOx) is related to the synthesis of glu-cosinolates in Brassicaceae and is an intermediate of the Trp-dependent biosynthesis of in-dole-3-acetic acid (IAA), sharing both oxime and IAA indolic structure. Furthermore, it has been well described that the IAA fits and interacts at the active centre of the horseradish peroxidase (POD). Indeed, the reaction of POD with IAA has been suggested as an onco¬logical therapy to produce toxic species. Throughout the first chapter of this work, several enzymes and oximes including IAOx were tested for NO production, by optimising the technical requirements and reac-tion conditions for its detection and measurement. As a result, it was demonstrated the NO production in vitro after IAOx oxidation catalysed by POD, as well as the important role of the superoxide radical (O2−) in this reaction. Moreover, it was shown that O2− and flavins significantly increased the production of NO, while oxygen (O2) and O2− depletion reduced it. Besides, it was assessed that the IAOx acted as a substrate for the mouse enzyme iNOS, producing significant amounts of NO. Finally, considering the results obtained, a new hy-pothesis for the NO oxidative production in plants was suggested, named as Mechanism for the Construction of an Analog of Nitric Oxide Synthase (MECANOS). In the second chapter, the effects of IAOx exposition in Arabidopsis thaliana wild-type (WT) plants, as well as its accumulation in sur1.1 mutants were analysed in plants growing in NH4+ as the sole N source. Afterwards, these plants showed the typical super-root phenotype, that shares characteristics with that of NO-exposed plants and has been described as a consequence of IAA accumulation; although it has been demonstrated that the IAOx phenotype differs from that after IAA exposition. Furthermore, there were report¬ed higher levels of NO in A. thaliana roots exposed to IAOx than in the control or in IAA exposed plants, by the DAF-2 DA sensing probe. Even more, both WT plants exposed to IAOx and sur1.1 mutants showed increased levels of internal IAA than the untreated con¬trol. Finally, the analysis of the genetic expression of several A. thaliana peroxidases showed that both WT plants externally exposed to IAOx and sur1.1 mutants downregulated these extracellular or intracellular enzymes, respectively, proving that NO production by IAOx was tightly transcriptionally regulated. Altogether, the in vivo effects of IAOx in A. thaliana were demonstrated to be consequence of an accumulation of IAA and an increase in NO. In the third chapter, it was addressed the analysis of IAOx effects on the molecular contents of A. thaliana cell cultures. Successfully, a total of 26 molecules was detected by GC-MS and catalogued. Subsequently, a labelling process prior to a Principal Component Analysis (PCA) confirmed that the reduction in Trp contents observed in cells was related to IAOx and NO donor S-nitrosoglutathione (GSNO) treatments and, therefore, with NO. Even more, the differences between the molecular contents of cells treated with IAOx and GSNO were mainly explained by those in benzenepropanoic acid, a member of the phenyl¬propanoids family highly associated with IAA levels. Consequently, IAOx addition indeed produced NO within A. thaliana cells, and IAOx either can serve as IAA source or disrupt the homeostasis. All in all, throughout the pages of this work, there are provided several in vitro and in vivo pieces of evidence to affirm that the oxidation of IAOx produces NO, together with sev-eral proofs of NO effects on root organogenesis, gene expression, and molecular contents in A. thaliana plants.