Biology subjects

Liebsch, D.

Publications and source records attributed to Liebsch, D..

2 recordsLinked to original sources

bioRxiv2025

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

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

plant biology↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗