No abstract

Energy homeostasis is vital to all living organisms. In eukaryotes, this process is controlled by fuel gauging protein kinases: AMP-activated kinase in mammals, Sucrose Non-Fermenting1 (SNF1) in yeast (Saccharomyces cerevisiae), and SNF1-related kinase1 (SnRK1) in plants. These kinases are highly conserved in structure and function and (according to this paradigm) operate as heterotrimeric complexes of catalytic-a and regulatory band g-subunits, responding to low cellular nucleotide charge. Here, we determined that the Arabidopsis (Arabidopsis thaliana) SnRK1 catalytic a-subunit has regulatory subunitindependent activity, which is consistent with default activation (and thus controlled repression), a strategy more generally used by plants. Low energy stress (caused by darkness, inhibited photosynthesis, or hypoxia) also triggers SnRK1a nuclear translocation, thereby controlling induced but not repressed target gene expression to replenish cellular energy for plant survival. The myristoylated and membrane-associated regulatory b-subunits restrict nuclear localization and inhibit target gene induction. Transgenic plants with forced SnRK1a-subunit localization consistently were affected in metabolic stress responses, but their analysis also revealed key roles for nuclear SnRK1 in leaf and root growth and development. Our findings suggest that plants have modified the ancient, highly conserved eukaryotic energy sensor to better fit their unique lifestyle and to more effectively cope with changing environmental conditions.

The SnRK1 kinases are key regulators of the plant energy balance, but how their activity is regulated by metabolic status is still unclear. While the heterotrimeric kinase complex is well conserved between plants, fungi and animals, plants appear to have modified its regulation to better fit their unique physiology and lifestyle. The SnRK1 kinases control metabolism, growth and development, and stress tolerance by direct phosphorylation of metabolic enzymes and regulatory proteins and by extensive transcriptional regulation. Diverse types of transcription factors have already been implicated, with a well-studied role for the heterodimerizing group C and group S1 bZIPs. SnRK1 is also part of a more elaborate metabolic and stress signaling network, which includes the TOR kinase and the ABAsignaling SnRK2 kinases.

Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1).Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches.We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10.Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.

The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here, we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labeling and cross-linking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with Class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.