Upstream regulators of the plant energy sensor SnRK1

Plants arguably are the most crucial organisms to support life on Earth because of their conversion of electro-magnetic energy (sunlight) into chemical energy (energy-rich carbohydrates) by photosynthesis and the associated release of oxygen. In addition, they produce innumerable renewable food, feed, fiber and fuel products and resources for mankind. Unlike most other organisms, autotrophic plants have a sessile (rooted) lifestyle. It is thus essential for plants to continuously monitor their environment and rapidly detect small fluctuations in light-, nutrient- and energy availability. Plants therefore have adopted multiple signaling mechanisms to detect and respond to these changes. A key role was identified for the SnRK1 kinase (Sucrose non-fermenting 1-related kinase 1), the plant homolog of the animal AMP-activated kinase, AMPK, and yeast Sucrose non-fermenting 1, SNF1. The conserved heterotrimeric protein kinase complex, like its opisthokont homologs, acts as a 'fuel gauge' and is activated when the plants' energy levels drop. This triggers a reprogramming of metabolism to an energy-saving and a survival mode by activating ATP producing pathways (catabolism) and repressing ATP consuming processes (anabolism). One of the consequences of SnRK1's central role in the maintenance of cellular energy homeostasis is that it directly or indirectly affects virtually every process in the plant, from general stress responses to key developmental transitions and growth. Understanding how exactly SnRK1 is regulated and how it affects its many downstream targets is thus crucial, both for our fundamental understanding of plant function and for the development of new strategies to increase plant stress tolerance and crop yields. In this work, we investigated Arabidopsis SnRK1 regulation using cellular assays with transient expression in leaf mesophyll protoplasts and mutant and transgenic lines. We more specifically tried to shed light on the molecular mechanisms behind SnRK1 complex formation and the extensive negative regulation of SnRK1 activity by (i) its regulatory subunits, (ii) catalase enzymes, and (iii) a novel plant-specific family of proteins, the SnRK1-Interacting Negative regulators or SKINs. First, we reviewed the present knowledge of SnRK1 structure, function and regulation, focusing on the most recent mechanistic insight. The strong conservation of this eukaryotic kinase complex enables the use of knowledge from yeast and animal systems. We also produced a 3D molecular model, based on homology with the AMPK complex, which gives insight in the plant complex' regulation and enables the generation of new working hypotheses. Secondly, we explored the mechanisms of SnRK1 activity regulation. Unlike animal AMPK and yeast SNF1, the plant kinase is not regulated by nucleotide charge (AMP/ATP ratio's). Instead, the kinase complex appears to be inhibited by sugar phosphates. Consistently, we found that the SnRK1α1 subunit is constitutively active. Diverse types of low energy stress trigger nuclear translocation of the SnRK1αsubunit and this translocation appears to be sufficient to induce target gene expression. The regulatory βsubunits act as negative regulators, at least in part by restricting nuclear localization. Transgenic plants with altered a subunit localization are affected in both development and metabolic stress responses, revealing new SnRK1 functions. The inhibitory function of the βsubunits depends on a poorly characterized N-terminal domain that appears to be regulated by post-translational modification in addition to N-terminal myristoylation. Thirdly, we identified the Arabidopsis H2O2-hydrolyzing catalase (CAT) enzyme as novel negative regulator of SnRK1 signaling. Catalases are typically localized in peroxisomes, but we found that they also partly localize in the cytoplasm, where they interact with and inhibit SnRK1αby local dismutation of H2O2. This appears to involve alternative splicing of the CAT peroxisomal targeting sequences in addition to cytosolic retention. Consistently, H2O2appears to act as a SnRK1 potentiator at low concentrations, possibly by direct redox-regulation of conserved Cys residues in the SnRK1αsubunits. Finally, we showed that a class of negative upstream regulators of SnRK1 signaling that was recently identified in rice (the SKIN proteins) is functionally conserved in Arabidopsis. The Arabidopsis SKIN orthologs and closely related AtOXS3-family proteins (identified in a screen for proteins that increase oxidative stress tolerance) co-localize and interact with the SnRK1αsubunit. Similar to the rice proteins, they appear to repress SnRK1 signaling by stimulating translocation of the αsubunit out of the nucleus. In conclusion, our results indicate that plants have tuned an ancient and highly conserved key regulatory pathway according to their preferred negative regulation strategy to better fit their autotrophic and sessile lifestyle and more efficiently cope with rapidly changing environmental conditions. In this work, we have identified several novel negative regulators and suggest directions for future research to further elucidate the molecular mechanisms involved.status: publishe