Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues

David B Medeiros; Stéphanie Arrivault; Jessica Alpers; Alisdair R Fernie; Fayezeh Aarabi

doi:10.21769/BioProtoc.3399

Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues

Bio Protoc. 2019 Oct 20;9(20):e3399. doi: 10.21769/BioProtoc.3399.

Authors

David B Medeiros¹, Stéphanie Arrivault¹, Jessica Alpers¹, Alisdair R Fernie¹, Fayezeh Aarabi¹

Affiliation

¹ Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476, Potsdam-Golm, Germany.

Abstract

The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of in-vivo metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.

Keywords: Compartment-specific markers; In-vivo metabolite distribution; Non-aqueous fractionation (NAF).