Quantification of SnRK1.1 response through analysis of its intracellular distribution

SnRK1 response and cellular energy status are evaluated using a novel N/ER index, which reflects changes in the intracellular distribution of its catalytic subunit, SnRK1.1. Maintaining energy homeostasis is a major challenge for plants facing changes in growth conditions. The Sucrose non-Fermenting 1 (SNF1) Related Kinase 1 (SnRK1) complex is a central player in securing cell energy homeostasis. The α subunit of this complex, also known as SnRK1.1, is a protein kinase that plays a critical role in sensing energy status and coordinating metabolic reprogramming to counter any energy imbalance. The discovery of a dual and dynamic intracellular distribution of SnRK1.1 suggests that its activity and function might be regulated by spatiotemporal changes. To investigate the link between the spatiotemporal localization of SnRK1.1 and SnRK1 response, we developed a protocol to quantify its intracellular distribution. We conceptualized and defined a new parameter, the N/ER index, which quantifies changes in distribution between nuclear and non-nuclear SnRK1.1 fractions. Using fluorescence confocal images acquired along the z-axis in plants expressing SnRK1.1-eGFP, and the open-source software Fiji/ImageJ, we calculated this parameter under control conditions and in plants treated with DCMU, a well-known trigger of SnRK1 response. These results showed that changes in SnRK1.1 intracellular localization constitute a major mechanistic step in the SnRK1-mediated response to restore energy homeostasis in planta. In addition, we establish the compatibility of our robust and simple method with a commercial software-based approach with different segmentation and quantification tools. Finally, our work demonstrates that N/ER index serves as a readout of SnRK1 response cell energy levels.