Characterizing the role of Arabidopsis nitrobindin in nitric oxide signaling

Nitric oxide (NO) is a versatile signal molecule that plays a crucial role during the entire lifespan of the plant. Even research has been focus on NO in plants for many years, a clear picture of NO signaling is still far from been achieved, neither in NO biosynthesis nor in downstream signaling of NO. The best characterized NO biosynthesis in plants is nitrite-dependent pathway via nitrate reductase (NR) mainly catalyzed by NR. However, it is believed that the existence of alternative enzymes or proteins to produce NO from nitrite in additional to NR under physiology conditions. Plants perceive and transduce NO signal transduction either through the post transcriptional modification of the proteins that can be directly targeted by NO or indirectly via NO receptor to activate second messengers. It is believed that, like in animal cells, NO signal can also be relay by the NO receptors. Nitrobindin is an unknown function heme-protein in Arabidopsis with NO binding properties. Based on its structural and functional similarity to the NPs, which play as NO transporter in insects and for which an NO-producing activity been recently reported, we assumed that nitrobindin could be involved in plant NO signaling either as NO producer or as NO receptor. In this study, the NO emission from nitrite by recombinant nitrobindin in vitro and the observation that NO emission was reduced in AtNB-RNAi plants during plant HR supported the initial hypothesis regarding the existence of an alternative route for nitrite-dependent NO production. Nevertheless, it was concluded that nitrobindin is not an NO producer in plants based on its low NO-producing activity of nitrobindin in comparison with NR and the absence of signal detected by NO electrode. A massive change in gene expression that analyzed by RNA-Seq due to the silencing of AtNB in Arabidopsis during plant NO fumigation indicated that nitrobindin functions as a NO transducer involved in NO mediate signal transduction pathway in plants. This was further supported by showing the genes that modulated by nitrobindin are also involved in NO signaling in plants. It was assumed that nitrobindin could function as NO transducer through its interaction with other protein(s) since it is unlikely a transcription factor because it does not contain any DNA-binding domain. A potential interactor of nitrobindin, NodGS, has been identified by TAP/MS platform specific upon the stimulation of NO. However, the interaction between NodGS and nitrobindin needs more evidences to confirm, and their role in NO signaling in plant needs further investigation.