Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

Abstract

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

The SnRK1 Kinase as Central Mediator of Energy Signaling between Different Organelles

“…However, being a protein kinase, direct targets of SnRK1 need to be measured by phosphoproteomics. To this end, we applied a quantitative phosphoproteomic approach combined with proteomics and metabolomics to identify in vivo targets of SnRK1 and to uncover the related metabolic reprograming using SnRK1 mutants under energy starvation (Nukarinen et al, 2016). This study revealed hundreds of changed phosphoproteins and a very pronounced SnRK1-dependent reprogramming of metabolism including sugars.…”

Recent Discoveries on the Role of TOR (Target of Rapamycin) Signaling in Translation in Plants

“…Recent work by Nukarinen et al (2016) has indicated that activation of SnRK1 is implicated in repression of protein synthesis (as main energy consumer) and implicated SnRK1 in regulation of photosynthesis (as main energy source); Weckwerth and colleagues found that ribosomal protein S6 was highly phosphorylated in Arabidopsis SnRK-deficient plants at , indicating that these phosphorylation sites are responsive to TOR activation (Nukarinen et al, 2016).…”

“…A similar antagonistic relationship between the AMPK plant ortholog SnRK1 (Suc nonfermenting 1-related Kinase 1) and TOR-related signaling pathways in response to changing nutritional and energy conditions was suggested in Arabidopsis Broeckx et al, 2016;Baena-González and Hanson, 2017). In mammals, being upstream of TOR, SnRK1 may inhibit TOR activity via direct interaction and phosphorylation of upstream components of TOR signaling and Raptor-the regulatory subunit of the TOR complex (Nukarinen et al, 2016)-possibly leading to complex disassembly (Hughes Hallett et al, 2015). Accordingly, in plants SnRK1 can interact and phosphorylate Raptor (Nietzsche et al, 2016;Nukarinen et al, 2016).…”

“…In mammals, being upstream of TOR, SnRK1 may inhibit TOR activity via direct interaction and phosphorylation of upstream components of TOR signaling and Raptor-the regulatory subunit of the TOR complex (Nukarinen et al, 2016)-possibly leading to complex disassembly (Hughes Hallett et al, 2015). Accordingly, in plants SnRK1 can interact and phosphorylate Raptor (Nietzsche et al, 2016;Nukarinen et al, 2016).…”

MRF Family Genes Are Involved in Translation Control, Especially under Energy-Deficient Conditions, and Their Expression and Functions Are Modulated by the TOR Signaling Pathway

“…Similarly, a tight correlation between translation activity and cellular sugar levels has been observed in plants (Pal et al, 2013;Lastdrager et al, 2014). The key players of plant energy signaling are sucrose non-fermenting 1-related protein kinase 1 (SnRK1) and TOR, which act in an antagonistic crosstalk in the plant's response to energy deprivation (Lastdrager et al, 2014;Mair et al, 2015;Nukarinen et al, 2016). TOR silencing mimics energy starvation conditions and activates catabolic processes and autophagy while repressing global translation (Deprost et al, 2007;Moreau et al, 2012;Ren et al, 2012;Caldana et al, 2013;Xiong et al, 2013).…”