Changes in intracellular distribution of SnRK1.1 in Arabidopsis thaliana

BRUGANARA, CANDELA; GIARRATANA, GIANINA; BULTRI, JULIÁN; HITA, FRANCISCO; PANOZZO ZENERE, ESTEBAN; BLANCO, NICOLÁS E.

Rosario

Congreso; SAIB 2023; 2023

The energy homeostasis is one of the most important process in all organisms. In the nature, differentmechanisms exist to maintain this balance correctly. For instance, when energy levels decrease due toenvironmental stresses, such as drought, extended darkness, and/or nutrient deficiency, a conservedkinase protein complex named AMPK/SNF1/SnRK1 has been identified as central component to restoreenergy balance in eukaryotes. In plants, controlling energy homeostasis presents a significantchallenge during adaptation to unfavourable growth conditions. Therefore, the SnRK1 complex serves asthe central integrator of energy imbalance signals and coordinator of the responses. The catalyticsubunit complex of SnRK1, known as SnRK1.1, exhibits dual and dynamic intracellular localization, beingpresent in the nucleus and endoplasmic reticulum (ER). Studies on the intracellular localization of SnRK1suggest that a fraction associated with the ER plays a crucial in sensing energy imbalances (Blanco etal., 2019). Based on this, we propose that changes in intracellular distribution of SnRK1.1 are linked to thesense to energy status and orchestration of the energy restoration. We developed a protocol that allowus to quantify the intracellular distribution of SnRK1.1 in nucleus and ER. Using laser scanning confocalmicroscopy (LSCM), we took images along z-axis of leaves under study and conducted an analysis withImageJ-Fiji to calculate de intensity fluorescence in different regions of interest (ROI), including one fornucleus and another for ER. The obtained ratio between these values provide us a parameter or index ofSnRK1.1 intracellular distribution. To validate this approach and highlight the effect of energy imbalanceon the SnRK1.1, we tested 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and Metformin (MET), twocompounds that perturb cell energy homeostasis. DCMU is known as a photosynthesis inhibitor thatblocks the plastoquinone binding site of photosystem II, decoupling the electron flow from lightabsorption. MET is a first-line treatment for type 2 Diabetes (T2D) and activates AMPK (mammalianorthologue of SnRK1 (Li et al., 2018)). Treatments were conducted in A. thaliana lines expressingSnRK1.1-fused to GFP and a second fluorescent organelle marker. SnRK1.1 intracellular distribution wasanalysed at different time points. In line with previous results, significative changes were observedbetween the intracellular SnRK1.1 fraction, with an augment in the population of this kinase localized inthe nucleus. Moreover, the compounds exhibited different response times and magnitudes. Theseresults, along with the physiological aspects of the treatments on lines with different SnRK1.1 content, arediscussed in the presentation.Based on our findings, we propose changes in the catalytic subunit distribution of the AMPK/SNF1/SnRK1complexes, which are common in the signalling mechanisms involved in cell energy homeostasis.