Studies of vacuolar transporters controlling nutrient storage and ion detoxification in Arabidopsis thaliana

• Applicant: Dr. Melanie Krebs
• Subject Area: Plant Physiology ; Plant Cell and Developmental Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316850664

• Final Report Year: 2021

Final Report Abstract

The acidification of plant endomembrane compartments is of great importance for various physiological processes as the activity of secondary active transporters critically depends on the transmembrane proton gradient which is established by vacuolar-type H+-ATPases (V- ATPases) at the trans-Golgi network/early endosome (TGN/EE) and the vacuole as well as by tonoplast-intrinsic H+-pyrophosphatases. Secondary active anion transporters of the Chloride Channel (CLC) family have been shown to be implicated in vacuolar chloride uptake thereby contributing to salt tolerance as well as they have been shown to be required for vacuolar storage of nitrate. Moreover TGN/EE localised CLCs have been proposed to support TGN/EE acidification by providing counterion uptake at the TGN/EE which could act as an electronic shunt that supports V-ATPase mediated acidification. In this project we aimed to get deeper knowledge about the individual physiological roles of V-ATPases and members of the CLC family situated at the TGN/EE and the vacuole. Furthermore, we intended to understand how V-ATPase mediated H+-fluxes and CLC-mediated anion fluxed have to be coordinated during nutrient storage and salt stress. We combined molecular physiology approaches, electrophysiology techniques, genetics and the use of pH sensitive dyes and genetically encoded pH indicators to reveal the function of V-ATPases and CLCs at the vacuole and TGN/EE. The project resulted in two important findings. (1) Function of CLCs at the TGN/EE is essential for plants and is required to prevent hyper-acidification of the Golgi stack. (2) CLCa function at the vacuolar membrane is critical for control of vacuolar pH. Furthermore, the ratio of nitrate and proton exchange mediated by CLCa is critical for plants to balance between the adjustment of osmotic pressure and nitrate assimilation which is an important agronomic trait. Moreover, our results indicate that there is indeed physical interaction between the V-ATPase subunit VHA-a3 and CLCa and CLCb, respectively. In addition, we have acquired evidence for a feedback regulation on the transcriptional level, as expression of CLCa and CLCb is decreased in the vha-a2 vha-a3 mutant, which is deficient for vacuolar V-ATPase activity. Taken together our results underline that activities of V-ATPases and CLCs at the TGN/EE and vacuole require to be coordinated in order to control endomembrane pH and anion homeostasis in plants.

Publications

• (2018). Green light for quantitative live-cell imaging in plants. J. Cell Sci. 131: jcs209270
  Grossmann, G., Krebs, M., Maizel, A., Stahl, Y., Vermeer, J.E.M., and Ott, T.
  
• (2021), An integrative view on vacuolar pH‐homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation. The Plant Journal
  Holzheu, P., Krebs, M., Larasati, C., Schumacher, K. and Kummer, U.