A new method to quantify the spatiotemporal localization of SnRK1.1

Brugnara, C. B.; Diaz, M. C.; Bultri, J.; Liebsch, D.; Hita, F.; Aguilar, D.; Fusari, C. M.; Dengjel, J.; Levi, V.; Blanco, N. E.

doi:10.1101/2025.04.14.648863

bioRxiv · 10.1101/2025.04.14.648863

A new method to quantify the spatiotemporal localization of SnRK1.1

Brugnara, C. B.·Diaz, M. C.·Bultri, J.·Liebsch, D.·Hita, F.·Aguilar, D.·Fusari, C. M.·Dengjel, J.·Levi, V.·Blanco, N. E.

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Abstract

Maintaining energy homeostasis is a major challenge for plants in the current context of climate change. The Sucrose-non fermenting 1 (SNF1)-related kinase 1 (SnRK1) complex, a member of the SNF1-AMP-activated protein kinase (AMPK)-SnRK1 family of kinase complexes, is a central player in the regulation of cell energy homeostasis. The -subunit of the complex, which possesses kinase activity and is known as SnRK1.1 or KIN10, plays a 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 the activity and function of SnRK1 might be regulated by spatiotemporal changes. To investigate the spatiotemporal distribution of SnRK1.1, we developed a protocol to quantify its intracellular distribution using fluorescence confocal images acquired along the z-axis in plants expressing SnRK1.1-eGFP. Using the open-source software Fiji/ImageJ, we calculated the ratio between nuclear and non-nuclear SnRK1.1 fractions and defined this as the N/ER index. We validated our method by analyzing the response of SnRK1.1 to photosynthesis inhibition by DCMU, including changes in protein levels and phosphorylation status. In addition, comparison with results obtained using a commercial software-based approach confirmed the compatibility of the N/ER index with different segmentation and quantification tools. Originally designed for leaf tissue images, this protocol can be broadly applied to assess the role of intracellular spatiotemporal changes in a wide range of kinases or fluorescently tagged recombinant proteins. Finally, SnRK1.1 intracellular distribution may also serve as a proxy to assess changes in cellular energy status. One sentence summaryNew method to track SnRK1.1 distribution and changes in plant cell energy status

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Brugnara, C. B., Diaz, M. C., Bultri, J., Liebsch, D., Hita, F., Aguilar, D., Fusari, C. M., Dengjel, J., Levi, V., Blanco, N. E.. 2025-04-19. A new method to quantify the spatiotemporal localization of SnRK1.1. https://doi.org/10.1101/2025.04.14.648863

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