A Nitrogen-specific Interactome Analysis Sheds Light on the Role of the SnRK1 and TOR Kinases inPlant Nitrogen Signaling - part2 kinase assay

ABSTRACT: Nitrogen (N) is of utmost importance for plant growth and development. Multiple studies have shown that N signaling is tightly coupled with carbon (C) levels, but the interplay between C/N metabolism and growth remains largely an enigma. Nonetheless, the protein kinases sucrose non-fermenting 1 (SNF1)-related kinase 1 (SnRK1) and target of rapamycin (TOR), two ancient central metabolic regulators, are emerging as key integrators that link C/N status with growth. Despite their pivotal importance, the exact mechanisms behind the sensing of N status and its integration with C availability to drive metabolic decisions are largely unknown. Especially for SnRK1 it is not clear how this kinase responds to altered N levels. Therefore, we first monitored N-dependent SnRK1 kinase activity at high resolution with our in vivo separation of phase-based activity reporter of kinase (SPARK), revealing a contrasting N-dependency in shoot and root tissues. Next, using affinity purification (AP) and proximity labeling (PL) coupled to mass spectrometry (MS) experiments, we constructed an N-specific SnRK1 and TOR interactome in Arabidopsis thaliana cell cultures during N-starved and  repleted growth conditions. To broaden our understanding of the N-dependency of the TOR/SnRK1 signaling pathway, the resulting network was compared to corresponding C-related networks, identifying a large number of novel, N-specific interactors. Moreover, through integration of N dependent transcriptome and phosphoproteome data, we were able to pinpoint additional N dependent network components, revealing SnRK1 regulatory proteins that might function at the crosstalk of C/N signaling. Finally, confirmation of known and identification of novel interactors, such as IRE1A and RAB18, indicate that the endoplasmic reticulum functions as a key regulatory hub for TOR and SnRK1 via integration of C/N signaling. Part2, kinase assay on RAB18, PANK2, SEC12 and NLP8.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Jelle Van Leene  

LAB HEAD: Geert De

PROVIDER: PXD054002 | Pride | 2024-11-12

REPOSITORIES: Pride