RESUMEN
NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Nitratos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismoRESUMEN
Both the phosphorylation and dephosphorylation of plant proteins is involved in multiple biological processes, especially in regard to signal transduction. The identification of phosphopeptides from MS (mass spectrometry)-based methods and their subsequent quantification play an important role in plant phosphoproteomics analysis. Phosphopeptide(s) identification and label-free quantification can determine dynamic changes of phosphorylation events in plants. Both MaxQuant and Proteome Discoverer are professional software tools used to identify and quantify large-scale MS-based phosphoproteomic data. This chapter gives a detailed workflow of MaxQuant and Proteome Discoverer software to analyze large amounts of phosphoproteomic-related MS data for the identification and quantification of label-free plant phosphopeptides.
